Related Applications

[001] This application claims priority to U.S. Provisional Application No. 62/277,438, filed on January 11, 2016; U.S. Provisional Application No. 62/291,470, filed on February 4, 2016; U.S. Provisional Application No. 62/291,461, filed on February 4, 2016; U.S.

Provisional Application No. 62/291,468, filed on February 4, 2016; PCT Application No. PCT/US2016/020530, filed on March 2, 2016; PCT Application No. PCT/US2016/050836, filed on September 8, 2016; U.S. Provisional Application No. 62/347,508, filed on June 8, 2016; U.S. Provisional Application No. 62/354,682, filed on June 24, 2016; U.S. Provisional Application No. 62/362,954, filed on July 15, 2016; U.S. Provisional Application No.

62/385,235, filed on September 8, 2016; U.S. Provisional Application No. 62/423, 170, filed on November 16, 2016; U.S. Provisional Application No. 62/439,871, filed on December 28, 2016; U.S. Provisional Application No. 62/347,576, filed on June 8, 2016; U.S. Provisional Application No. 62/348,620, filed on June 10, 2016; PCT Application No.

PCT/US2016/039444, filed on June 24, 2016; and PCT Application No. PCT/US2016/069052, filed on December 28, 2016, the entire contents of each of which are expressly incorporated herein by reference.

Background

[002] This disclosure relates to compositions and therapeutic methods for detoxifying deleterious molecules. In certain aspects, the disclosure relates to genetically engineered bacteria that are capable of inhibiting and/or metabolizing toxic molecules, metabolites, or other deleterious molecules. In certain aspects, the compositions and methods disclosed herein may be used to detoxify chemotherapeutic drugs or metabolites or byproducts thereof (e.g., 5-fluorouracil, irinotecan), nonsteroidal ant i- inflammatory drugs or metabolites or byproducts thereof (e.g., naproxen, indomethacin), or exogenous toxic molecules (e.g. , lead, dioxin). Genetically engineered bacteria that are capable of detoxifying endogenous toxic molecules have been described elsewhere, for example, see PCT/US 2015/64140 and

PCT/US2016/34200 (detoxifying excess ammonia); PCT/US2016/32562 and

PCT/US2016/062369 (detoxifying excess phenylalanine); PCT/US2016/37098 (detoxifying excess branch chain amino acids); PCT/US2016/044922 (detoxifying excess propionate); PCT/US2016/049781 (detoxifying excess oxalate), the entire contents of which are expressly incorporated herein by reference. [003] The mammalian body routinely encounters toxic substances, including environmental toxic molecules, therapeutic drugs, and endogenously produced molecules. At low levels, some toxic substances can be detoxified naturally by the body. For example, naturally occurring hepatic enzymes are capable of detoxifying ammonia, a toxic substance that can be endogenously produced during amino acid biosynthesis, and alcohol, a toxic substance that can be exogenously ingested. However, many toxic substances cannot be detoxified naturally by the body, particularly when they are present at high levels. These toxic substances can cause disastrous physiological effects, including death.

[004] Heavy metal poisoning can cause damage to the brain, lungs, kidneys, liver, and blood (Jaishankar et al., 2014). For example, copper poisoning is capable of causing gastrointestinal hemorrhaging, basal ganglia neurodegeneration, stroke, and death. Lead poisoning is capable of causing renal dysfunction, mental retardation, and death. Heavy metal poisoning can occur by exposure through the skin, ingestion, and/or inhalation, and young children are particularly vulnerable. Current therapies for heavy metal poisoning include natural and synthetic chelating agents such as metal binding proteins, metallothioneins, and small organic molecules. However, only certain heavy metals can be chelated by particular chelating agents, and the treatment window can be limited (Smith 2013 ; Sears 2013).

Furthermore, there is no support for the efficacy of chelation in chronic toxicity (Smith 2013).

[005] Chemotherapeutic drugs and nonsteroidal ant i- inflammatory drugs are administered for therapeutically beneficial effects, but those drugs and/or their metabolites or byproducts are also capable of causing deleterious effects. For example, chemotherapy is commonly associated with dose-limiting diarrhea, and chemotherapy-induced diarrhea is a notable cause of morbidity and mortality (Andreyev et al., 2014; Stein et al., 2010). Diarrhea occurs in about 50-80% of chemotherapy patients, particularly those treated with irinotecan, fluoropyrimidines, and/or 5-fluorouracil (Stein et al., 2010). Grade 3 diarrhea is severe enough to affect daily life, grade 4 diarrhea is life-threatening, and grade 5 diarrhea can cause death. Patients who experience chemotherapy-induced diarrhea may need to pause, adjust, or even discontinue chemotherapy, thereby interfering with or detracting from cancer treatment, which can impact survival (Stein et al., 2010). Likewise, nonsteroidal ant i- inflammatory drugs are also associated with dose-limiting diarrhea and gastrointestinal toxicity, which interfere with or detract from treatment (Scarpignato, 2008).

[006] Some medications, such as folic acid analogs, folinic acid, and leucovorin, are capable of reducing the general toxic effects on healthy cells caused by chemotherapeutic drugs. No pharmacological strategies effectively prevent radiotherapy-induced diarrhea (Andreyev et al, 2014). Suggested therapies to ameliorate diarrhea include activated charcoal, glutamine, celecoxib, racecadotril, and probiotics, but "evidence of efficacy is lacking for all" (Andreyev et aL., 2014). Glutamine, sucralfate, sulfasalazine, and octreotide have all been tested in trials, but none decreased or prevented diarrhea (Andreyev et al., 2014).

[007] Thus, there is significant unmet need for effective, reliable, and/or long-term treatment for disorders and conditions caused by toxic molecules, metabolites, and other deleterious molecules.

Summary

[008] The invention provides genetically engineered bacteria that are capable of detoxifying, inhibiting, and/or metabolizing deleterious molecules. In certain embodiments, the genetically engineered bacteria are capable of detoxifying toxic molecules, metabolites, or other deleterious molecules selectively in low-oxygen environments, e.g., the mammalian gut. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to reduce toxicity. In some embodiments, the toxic molecule also exerts therapeutically beneficial effects, e.g., cytotoxicity to cancer cells, and the genetically engineered bacteria detoxify the toxic molecule or its metabolite(s) or byproduct(s) after the therapeutically beneficial effects have been exerted. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders and conditions caused by toxic molecules, metabolites, and other deleterious molecules, e.g., chemotherapy- induced diarrhea and heavy metal poisoning.

[009] In some embodiments, the genetically engineered bacteria are capable of inhibiting, metabolizing, and/or detoxifying chemotherapeutic drugs or metabolites or byproducts thereof. In some embodiments, the genetically engineered bacteria detoxify the drug or metabolite or byproduct after the chemotherapeutic drug exerts its therapeutically beneficial effects, e.g., cytotoxicity in cancerous cells. In some embodiments, the genetically engineered bacteria are administered before, together with, and/or after administration of the chemotherapeutic drug. In some embodiments, the genetically engineered bacteria are capable of detoxifying, inhibiting, and/or metabolizing the drug or metabolite or byproduct, thereby reducing chemotherapy-induced diarrhea, reducing chemotherapy-induced toxicity, increasing chemotherapy dosage amount, increasing chemotherapy dosage frequency, and/or increasing chemotherapy efficacy. In some embodiments, the molecule to be detoxified is a

chemotherapeutic drug selected from irinotecan, methotrexate, an antimetabolite, gemcitabine, cytosine arabinoside, a fluoropyrimidine, fluoro uracil, capecitabine, tegafur- uracil, a multitargeted folinic acid antagonist, pemetrexed, raltitrexed, gemcitabine, a plant alkaloid, a vinca alkaloid, vincristine, vinorelbine, a epipodophyllotoxin, etoposide, a taxane, paclitaxel, docetaxel, a topoisomerase I inhibitor, a cytotoxic antibiotic, an anthracycline, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, an alkylating agent, cyclophosphamide, a platinum, cisplatin, carboplatin, oxaliplatin, nedaplatin, an antibody, ipilumumab, an antibody against VEGF, bevacizumab, a tyrosine-kinase inhibitor, an EGFR inhibitor, lapatinib, cetuximab, or a metabolite or byproduct of said chemotherapeutic drug, e.g. , SN-38.

[0010] In some embodiments, the engineered bacteria comprise gene sequence encoding one or more enzyme(s) capable of metabolizing or otherwise detoxifying a toxic or deleterious molecule. In some embodiments, the genetically engineered bacteria further comprises gene sequence encoding one or more enzyme(s) for the production of one or more ant i- inflammatory molecule(s). In some embodiments, the genetically engineered bacteria further comprise gene sequence for the production of one or more gut barrier enhancer molecule(s). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s). In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more antiinflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is an auxotroph. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s )is an auxotroph and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.

[0011] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate. In some embodiments, the payload is a small molecule that is capable of inhibiting methotrexate. In some embodiments, the payload is an enzyme that is capable of metabolizing methotrexate into non-toxic metabolites. In certain embodiments, the enzyme capable of metabolizing methotrexate is from a non-human species, e.g., a plant, bacterial, or other mammalian enzyme. In some embodiments, the enzyme is a synthetic or modified enzyme. In some embodiments, the genetically engineered bacteria comprise a gene encoding carboxypeptidase Gi (CPD Gi) and are capable of detoxifying methotrexate. In certain embodiments, the genetically engineered bacteria comprise a gene encoding Pseudomonas stutzeri CPD Gi (Chabner et ah, 1972). In some embodiments, the genetically engineered bacteria comprise a gene encoding carboxypeptidase G ₂ (CPD G ₂ ) and are capable of detoxifying methotrexate.

[0012] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and/or SN-38. In some embodiments, the payload is a small molecule that inhibits β-glucuronidase to prevent the conversion of irinotecan into the toxic metabolite SN-38. In some embodiments, the small molecule that inhibits β-glucuronidase is D-saccharic acid 1.4-lactone (SAL). In some embodiments, the payload is a small molecule that is capable of inhibiting SN-38. In some embodiments, the payload is an enzyme that is capable of metabolizing SN-38 into non-toxic metabolites. In some embodiments, the payload is an enzyme that is capable of glucuroniding SN-38, thereby converting it into non-toxic SN-38G. In certain embodiments, the enzyme is from a non- human species, e.g., a plant, bacterial, or other mammalian enzyme. In some embodiments, the enzyme is a synthetic or modified enzyme. In some embodiments, the payload is a molecule that is capable of inhibiting or killing the commensal bacteria that produce β-glucuronidase. In some embodiments, the payload is a molecule that promotes changes to the intestinal microflora, e.g., enhancing the survival and/or proliferation of the genetically engineered bacteria, thereby outcompeting commensal bacteria that produce β- glucuronidase. In some embodiments, the genetically engineered bacteria comprise a gene encoding UDP-glucuronsyltransferase, which is capable of adding glucuronic acid to SN-38, and are capable of detoxifying SN-38.

[0013] In some embodiments, the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying NSAIDs or metabolites or byproducts thereof. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying one or more NSAIDs, e.g., naproxen. In some embodiments, the payload is a small molecule that is capable of inhibiting one or more NSAIDs, e.g., naproxen. In some embodiments, the payload is a proton pump inhibitor, and the genetically engineered bacteria are capable of ameliorating NSAID-induced intestinal damage. In some embodiments, the payload is an enzyme that is capable of metabolizing the NSAID into non-toxic metabolites. In certain embodiments, the enzyme capable of metabolizing the NSAID is from a non-human species, e.g. , a plant, bacterial, or other mammalian enzyme. In some embodiments, the enzyme is a synthetic or modified enzyme. B -glucuronidase inhibition can alleviate NSAID-induced enteropathy. In some embodiments, the genetically engineered bacteria comprise a gene encoding

glucuronosyltransferase and are capable of glucuronidating and detoxifying naproxen.

[0014] In some embodiments, the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying one or more heavy metals, thereby ameliorating one or more symptoms of heavy metal poisoning. In some embodiments, the genetically engineered bacteria of the invention are capable of ameliorating acute heavy metal poisoning and/or chronic heavy metal poisoning. In some embodiments, genetically engineered bacteria of the invention are capable of ameliorating one or more symptoms of aluminum poisoning, antimony poisoning, arsenic poisoning, barium poisoning, bismuth poisoning, cadmium poisoning, chromium poisoning, cobalt poisoning, copper poisoning, gold poisoning, iron poisoning, lead poisoning, lithium poisoning, manganese poisoning, mercury poisoning, nickel poisoning, phosphorous poisoning, platinum poisoning, selenium poisoning, silver poisoning, thallium poisoning, tin poisoning, and/or zinc poisoning.

[0015] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering a heavy metal. In some embodiments, the payload is a heavy metal chelator. In some embodiments, the payload is a plant phytochelatin. In certain embodiments, the payload is from a non-human species, e.g. , a plant, bacterial, or other mammalian molecule. In some embodiments, the payload is a synthetic or modified molecule. In some embodiments, the genetically engineered bacteria are capable of expressing plant phytochelatins, particularly on the surface of the bacteria. Thus, the genetically engineered bacteria of the invention are capable of binding to cadmium, thereby ameliorating one or more symptoms of cadmium poisoning.

[0016] In some embodiments, the payload is carboxypeptidase Gi (CPD Gi) or carboxypeptidase G ₂ (CPD G ₂ ). In some embodiments, the payload is D-saccharic acid 1, 4- lactone (SAL). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen.. In some embodiments, the payload is a proton pump inhibitor. In some embodiments, the payload is a heavy metal chelator. In some embodiments, the payload is a plant phytochelatin. In some embodiments, the payload is a short-chained fatty acid, e.g. butyrate, propionate, or acetate. In some embodiments, the payload is the enzyme Pseudomonas. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying a toxic or deleterious molecule and a gene or gene cassette for producing a short-chained fatty acid, e.g. butyrate, propionate, or acetate.

Brief Description of the Figures

[0017] FIG. 1A and IB depict schematics of the gene organization of exemplary bacteria of the invention. FIG. 1A depicts the gene organization of an exemplary recombinant bacterium of the invention comprising a butyrate synthetic cassette and a carboxypeptidase G2 cassette, wherein the butyrate synthetic cassette and the carboxypeptidase G2 cassette comprise a FNR-responsive promoter, and wherein FNR {e.g., a FNR dimer) binding to the FNR- responsive promoter induces the expression of the butyrate synthetic cassette and/or the carboxypeptidase G2 cassette, which leads to the production of butyrate and/or the production of carboxypeptidase G2 (CPD G2). The bacteria may optionally include an auxo trophy, e.g., deletion or mutation of thyA (Δ thyA; thymidine dependence) in the E. coli Nissle genome, such that thymidine must be supplied in the culture medium to support growth. FIG. IB depicts the gene organization of an exemplary recombinant bacterium of the invention comprising a butyrate synthetic cassette and a glucuronosyl transferase cassette, wherein the butyrate synthetic cassette and the glucuronosyl transferase cassette comprise a FNR- responsive promoter, and wherein FNR {e.g., a FNR dimer) binding to the FNR-responsive promoter induces the expression of the butyrate synthetic cassette and/or the glucuronosyl transferase cassette, which leads to the production of butyrate and/or the production of glucuronosyl transferase (GT). The bacteria may optionally include an auxotrophy, e.g., deletion or mutation of thyA (Δ thyA; thymidine dependence) in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth

[0018] FIG. 1C and FIG. ID depict the state of a non- limiting embodiment certain constructs of the invention under non-inducing (FIG. 1C) and inducing (FIG.1D) conditions. FIG. 1C depicts relatively low carboxypeptidase G2 (CPD G2) and glucuronosyl transferase (GT) production under aerobic conditions due to oxygen (0 ₂ ) preventing FNR from dimerizing and activating carboxypeptidase G2 (CPD G2) and/or glucuronosyl transferase (GT) gene expression. FIG. ID depicts upregulated carboxypeptidase G2 (CPD G2) and/or glucuronosyl transferase (GT) production under anaerobic conditions due to FNR dimerizing and inducing FNR promoter-mediated expression of the carboxypeptidase G2 (CPD G2) and/or glucuronosyl transferase (GT) (squiggled line above "Carboxypeptidase G2" and "Glucuronosyl transferase"). Arrows adjacent to a single rectangle, or cluster of rectangles, depict the promoter responsible for driving transcription (in the direction of the arrows) of such gene(s). Arrows above each rectangle depict the expression of each gene.

[0019] FIG. 2A, FIG. 2B, FIG. 2C, and FIG.2D depict schematics of a butyrate production pathway and schematics of different butyrate producing circuits. FIG. 2A depicts a metabolic pathway for butyrate production. FIG. 2B and FIG. 2C depict schematics of two different exemplary butyrate producing circuits, both under the control of a tetracycline inducible promoter. FIG. 2B depicts a bdc2 butyrate cassette under control of a tet promoter on a plasmid. A "bdc2 cassette" or "bdc2 butyrate cassette" refers to a butyrate producing cassette that comprises at least the following genes: bcd2, etfB3, etfA3, hbd, crt2, pbt, and buk genes. FIG. 2C depicts a ter butyrate cassette (ter gene replaces the bcd2, etfB3, and etfA3 genes) under control of a tet promoter on a plasmid. A "ter cassette" or "ter butyrate cassette" refers to a butyrate producing cassete that comprises at least the following genes: ter, thiAl, hbd, crt2, pbt, and buk genes. FIG. 2D depicts a schematic of a third exemplary butyrate gene cassette under the control of a tetracycline inducible promoter, specifically, a tesB butyrate cassette (ter gene is present and tesB gene replaces the pbt gene and the buk gene) under control of a tet promoter on a plasmid. A "tes or tesB cassette or "tes or tesB butyrate cassette" refers to a butyrate producing cassette that comprises at least ter, thiAl, hbd, crt2, and tesB genes. An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiAl, hbd, crt2, and tesB genes. In some embodiments, the tes or tesB cassette is under the control of an inducible promoter other than tetracycline. Exemplary inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

[0020] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F depict schematics of the gene organization of exemplary bacteria of the disclosure. FIG. 3A and FIG. 3B depict the gene organization of an exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions. FIG. 3A depicts relatively low butyrate production under aerobic conditions in which oxygen (0 ₂ ) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk; white boxes) are expressed. FIG. 3B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIG. 3C and FIG. 3D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 3C, in the absence of NO, the NsrR transcription factor (circle, "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) are expressed. In FIG. 3D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[0021] FIG. 3E and FIG. 3F depict the gene organization of an exemplary

recombinant bacterium of the invention and their induction in the presence of H ₂ O ₂ . In FIG. 3E, in the absence of H ₂ O ₂ , the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) are expressed. In FIG. 3F, in the presence of H ₂ O ₂ , the OxyR transcription factor interacts with H ₂ O ₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[0022] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F depict schematics of the gene organization of exemplary bacteria of the invention. FIG. 4A and FIG. 4B depict the gene organization of another exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions using a different butyrate circuit from that shown in FIG. 3. FIG. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (O ₂ ) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, and buk; white boxes) are expressed. FIG. 4B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.

[0023] FIG. 4C and FIG. 4D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 4C, in the absence of NO, the NsrR transcription factor (circle, "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk) are expressed. In FIG. 4D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. FIG. 4E and FIG. 4F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H ₂ O ₂ . In FIG. 4E, in the absence of H ₂ O ₂ , the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes {ter, thiAl, hbd, crt2, pbt, buk) are expressed. In FIG. 4F, in the presence of H ₂ O ₂ , the OxyR transcription factor interacts with H ₂ O ₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[0024] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F depict schematics of the gene organization of exemplary bacteria of the invention. FIG. 5A and FIG. 5B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (O ₂ ) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes {ter, thiAl, hbd, crt2, and tesB) are expressed. FIG. 5B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIG. 5C and FIG. 5D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 5C, in the absence of NO, the NsrR transcription factor ( "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes {ter, thiAl, hbd, crt2, tesB) are expressed. In FIG. 5D, in the presence of NO, the NsrR

transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. FIG. 5E and FIG. 5F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H ₂ O ₂ . In FIG. 5E, in the absence of H ₂ O ₂ , the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter.

Therefore, none of the butyrate biosynthesis enzymes {ter, thiAl, hbd, crt2, tesB) are expressed. In Fig. 5F, in the presence of H ₂ O ₂ , the OxyR transcription factor interacts with H ₂ O ₂ and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[0025] Fig. 6 depicts a bar graph showing butyrate production of butyrate producing strains of the invention. FIG. 6 shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen (designated as "02"), in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl Co A). Overnight cultures of cells were diluted 1: 100 in Lb and grown for 1.5 hours until early log phase was reached at which point anhydrous tet was added at a final concentration of lOOng/ml to induce plasmid expression. After 2 hours induction, cells were washed and resuspended in M9 minimal media containing 0.5% glucose at OD6oo=0.5. Samples were removed at indicated times and cells spun down. The supernatant was tested for butyrate production using LC-MS.

[0026] FIG. 7 depicts a bar graph showing butyrate production of butyrate producing strains of the invention. FIG. 7 shows butyrate production in strains comprising a tet-butyrate cassette having ter substitution (pLOGIC046) or the tesB substitution (ptb-buk deletion), demonstrating that the tesB substituted strain has greater butyrate production.

[0027] FIG. 8 depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. Strains depicted are BW25113 comprising a bcd- butyrate cassette, with or without a nuoB deletion, and BW25113 comprising a ter-butyrate cassette, with or without a nuoB deletion. Strains with deletion are labeled with nuoB. The nuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains. NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.

[0028] FIG. 9A, FIG. 9B, and FIG.9C depict schematics and graphs showing butyrate production of a butyrate producing circuit under the control of an FNR promoter. FIG. 9A depicts a schematic showing a butyrate producing circuit under the control of an FNR promoter. FIG. 9B depicts a bar graph of anaerobic induction of butyrate production. FNR- responsive promoters were fused to butyrate cassettes containing either the bed or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes. Cells were washed and resuspended in minimal media w/ 0.5% glucose and incubated microaerobically to monitor butyrate production over time. SYN-501 led to significant butyrate production under anaerobic conditions. FIG. 9C depicts SYN-501 in the presence and absence of glucose and oxygen in vitro. SYN-501 comprises pSClOl PydfZ-ter butyrate plasmid; SYN-500 comprises pSClOl PydfZ-bcd butyrate plasmid; SYN-506 comprises pSClOl nirB-bcd butyrate plasmid.

[0029] FIG. 10 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H ₂ 0, 100 mM butyrate in H ₂ 0, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H ₂ 0 (+) 200 mM butyrate.

[0030] FIG. 11 depicts a bar graph comparing butyrate concentrations produced in vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia butyricum MIYARISAN (a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain) under aerobic and anaerobic conditions at the indicated timepoints. The Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.

[0031] FIG. 12 depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid copies. Integrated butyrate strains, SYN1001 and SYN1002 (both integrated at the agal/rsml locus) gave comparable butyrate production to the plasmid strain SYN501.

[0032] FIG. 13A and FIG. 13B depicts the construction and gene organization of an exemplary plasmids. FIG. 13A depicts the construction and gene organization of an exemplary plasmids comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct). FIG. 13B depicts the construction and gene organization of another exemplary plasmid comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic046- nsrR- norB -butyrogenic gene cassette).

[0033] FIG. 14 depicts butyrate production using SYN001 + tet (control wild-type Nissle comprising no plasmid), SYN067 + tet (Nissle comprising the pLOGIC031 ATC- inducible butyrate plasmid), and SYN080 + tet (Nissle comprising the pLOGIC046 ATC- inducible butyrate plasmid). [0034] FIG. 15 depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB -butyrate construct (SYN133) or the pLogic046-nsrR- norB-butyrate construct (SYN145), which produce more butyrate as compared to wild-type Nissle (SYN001).

[0035] Fig. 16 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown Table 3 (Pfnrl-5). Different FNR-responsive promoters were used to create a library of anaerobic/low oxygen conditions inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+0 ₂ ) or anaerobic conditions (- 0 ₂ ). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays.

[0036] Fig. 17A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P _fnr s)- LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. Fig. 17B depicts FNR promoter activity as a function of β- galactosidase activity in SYN-PKU904. SYN-PKU904, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr. under anaerobic and/or low oxygen conditions . Fig. 17C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

[0037] Fig. 18 depicts a construct comprising FNRS24Y driven by the arabinose inducible promoter and araC oriented in the reverse direction.

[0038] FIG. 19A depicts an "Oxygen bypass switch" useful for aerobic pre-induction of a strain comprising one or proteins of interest (POI), e.g., one or more propionate catabolism enzyme(s) (POI1) and /or one or more transporter(s)/importer(s) and/or exporter(s) (POI2) under the control of a low oxygen FNR promoter in vitro in a culture vessel {e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active propionate catabolism enzyme(s) prior to administration. This can be done by pre-inducing the expression of these enzymes as the strains are propagated, {e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites. FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ, The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and preloaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo. In some embodiments, a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

[0039] FIG 19B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y. By using a ribosome binding site optimization strategy, the levels of Fnr expression can be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bio informatics tools for optimization of RBS are known in the art.

[0040] FIG. 19C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bio informatics tools for optimization of RBS are known in the art. In one strategy, arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of Pfnrs-POI constructs are maintained to allow for strong in vivo induction. [0041] Fig. 20 depicts the gene organization of an exemplary construct, comprising a cloned cloned protein of interest (POI) gene under the control of a Tet promoter sequence and a Tet repressor gene.

[0042] Fig. 21 depicts the gene organization of an exemplary construct comprising Lacl in reverse orientation, and a IPTG inducible promoter driving the expression of a protein of interest (POI). In some embodiments, this construct is useful for pre-induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. PheP expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is used in combination with a LAAD expression construct. In some

embodiments, the constructs PAL3 sequences which are the original sequence from

Photorhabdus chemiluminescens. In some embodiments, the PAL3 sequences are codon optimized for expression in E coli. In some embodiments, the construct is located on a plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct is employed in a biosafety system, such as the system shown in Fig. 36A, Fig. 36B, Fig. 36C, and Fig. 36D. In some embodiments, the construct is integrated into the genome at one or more locations described herein.

[0043] Figs. 22A-C depict schematics of non-limiting examples of constructs expressing a protein of interest (POI). Fig 22A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, this construct is used alone. In some embodiments, the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with other POI constructs, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).

[0044] Fig. 22B depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter. For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only

the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In a non- limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

[0045] Fig. 22C depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of an arabinose inducible promoter. The arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and the POI gene. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre- induction and pre-loading of one or more POI(s) prior to in vivo administration. In a non- limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

[0046] Fig. 23A depicts a schematic of the gene organization of a PssB promoter. The ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair. The PssB promoter was cloned in front of a LacZ reporter and beta- galactosidase activity was measured. Fig. 23B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1: 100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).

[0047] Fig. 24 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites.

[0048] Fig. 25 depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent.

[0049] Fig. 26 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post- administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

[0050] Fig. 27 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating.

[0051] Fig. 28 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (Mo As).

[0052] Figs. 29A-29C depict other non- limiting embodiments of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. Fig. 29A depicts an embodiment of heterologous gene expression in which, in the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (P _araBAD ), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. Fig. 29A also depicts another non- limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a

conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell.

[0053] Fig. 29B depicts a non-limiting embodiment of the disclosure, where an antitoxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit.

[0054] Fig. 29C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.

[0055] Fig. 30 depicts one non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[0056] Fig. 31 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated

conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.

[0057] Fig. 32 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death.

[0058] Fig. 33 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[0059] Fig. 34 depicts a one non- limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti-toxin decays completely, and the cell dies.

[0060] Fig. 35 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al, 2015.

[0061] Figs. 36A-36D depict schematics of non- limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (Fig. 36A and Fig. 36B), which also contains a chromosomal component (shown in Fig. 36C and Fig. 36D). The Biosafety Plasmid System Vector comprises Kid Toxin and R6K minimal ori, dapA (Fig. 36A) and thyA (Fig. 36B) and promoter elements driving expression of these components. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, in which PAL3 and/or PheP and/or LAAD are expressed from an inducible or constitutive promoter. Fig. 36C and Fig. 36D depict schematics of the gene organization of the chromosomal component of a biosafety system. Fig. 36C depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter. Fig. 36D depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter. If the plasmid containing the functional DapA is used (as shown in Fig. 36A), then the chromosomal constructs shown in Fig. 36C and Fig. 36D are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in Fig. 36B), then the chromosomal constructs shown in Fig. 36C and Fig. 36D are knocked into the ThyA locus. In this system, the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.

[0062] Fig. 37 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[0063] Fig. 38 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N- terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.

[0064] Fig. 39 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP- binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[0065] Fig. 40 depicts a schematic of the outer and inner membranes of a gram- negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the

extracellular space, e.g. , therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g. , degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.

[0066] Fig. 41 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons).

[0067] Fig. 42A, Fig. 42B, and Fig. 42C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide of interest, such as, PAL and/or LAAD, is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (Fig. 42A and Fig. 42B) or a Tet-inducible promoter (Fig. 42C). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR- inducible promoter), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct, as shown in Fig. 42B and Fig. 42C.

[0068] Fig. 43A and Fig. 43B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpl. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a Tet promoter (Fig. 43A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, Fig. 43B), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose.

[0069] Fig. 44A depicts a schematic diagram of a wild-type clbA construct. Fig. 44B depicts a schematic diagram of a clbA knockout construct.

[0070] Fig. 45 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7.

Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animal disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen.

[0071] Figs. 46A, B, C, D, and E depict a schematic of non- limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. Fig. 46A depicts the parameters for starter culture 1 (SCI): loop full - glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm. Fig. 46B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SCI, duration 1.5 hours, temperature 37° C, shaking at 250 rpm. Fig. 46C depicts the parameters for the production bioreactor: inoculum - SC2, temperature 37° C, pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300- 1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours. Fig. 46D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS. Fig. 46E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.

[0072] FIG. 47 A and FIG. 47B depict diagrams of bacterial tryptophan metabolism pathways. FIG. 47A depicts a schematic of the bacterial tryptophan metabolism, as described, e.g. , in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase

(ECl .1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2-monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0). The dotted lines ( ) indicate a spontaneous reaction. FIG. 47B

Depicts a schematic of tryptophan derived pathways. Known AHR agonists are with asterisk. Abbreviations are as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole-3- acetaldehyde; IAA: Indole- 3 -acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA: Indole- 3-pyruvic acid; IAM: Indole- 3 -acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3- acetonitrile; N-formyl Kyn: N-formylkynurenine;; Kyn: Kynurenine; KynA: Kynurenic acid; I3C: Indole-3-carbinol; IAld: Indole- 3 -aldehyde; DIM: 3,3'-Diindolylmethane; ICZ:

Indolo(3,2-b)carbazole. Enzymes are numbered as follows: 1. EC 1.13.11.11 (Tdo2, Bna2), EC

I .13.11.11 (Ido l); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4. EC 1.2.1.3 (ladl), EC 1.2.3.7 (Aao l); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclbl, Cclb2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125 (CYP79B2, CYP79B3); 9. EC 1.4.3.2 (StaO), EC 2.6.1.27 (Aro9, aspC), EC 2.6.1.99 (Taal), EC 1.4.1.19 (TrpDH); 10. EC 1.13.12.3 (laaM);

I I . EC 4.1.1.74 (IpdC); 12. EC 1.14.13.168 (Yuc2); 13. EC 3.5.1.4 (IaaH); 14. EC 3.5.5.1. (Nitl); 15. EC 4.2.1.84 (Nitl); 16. EC 4.99.1.6 (CYP71A13); 17. EC 3.2.1.147 (Pen2). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIGs. 47A and 47B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIGs. 47A and 47B. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments the one or more cassettes are under the control of inducible promoters which are induced under low- oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[0073] FIG. 48 shows a schematic depicting an exemplary Tryptophan circuit.

Tryptophan is produced from the Chorismate precursor through expression of the trpE, trpG-D (also referred to as trpD), trpC-F (also referred to as trpC), trpB and trpA genes. Optional knockout of the tryptophan Repressor trpR is also depicted. Optional production of the

Chorismate precursor through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. All of these genes are optionally expressed from an inducible promoter, e.g. , a FNR- inducible promoter. The bacteria may also include an auxotrophy, e.g. , deletion of thyA (Δ thyA; thymidine dependence). The bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan. Non limiting example of a bacterial strain is listed.

[0074] FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG. 49E, FIG. 49F, FIG. 49G, and FIG. 49H depict schematics of non- limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included. FIG. 49A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. The optional circuits for tryptophan production are as depicted and described in FIG. 48. FIG. 49B depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan. FIG. 49C depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan. FIG. 49D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan. FIG. 49E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan. FIG. 49F depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan. FIG. 49G depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan. FIG. 49H depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3-carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. The genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g. , from Arabidopsis thaliana) under the control of an inducible promoter, e.g. an FNR promoter. The engineered bacterium shown in any of FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG. 49E, FIG. 49F, FIG. 49G and FIG. 49H may also have an auxo trophy, e.g., in one example, the thy A gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.

[0075] FIG. 50A, FIG. 50B, FIG. 50C, FIG. 50D, and FIG. 50E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole- 3 -acetic acid. In FIG. 50A, the optional circuits for tryptophan production are as depicted and described in FIG. 41. In FIG. 50B the optional circuits for tryptophan production are as depicted and described in FIG. 41. In FIG. 50C the optional circuits for tryptophan production are as depicted and described in FIG. 41. In FIG. 50D the optional circuits for tryptophan production are as depicted and described in FIG. 41. In FIG. 50E the optional circuits for tryptophan production are as depicted and described in FIG. 41.

[0076] FIG. 51A and FIG 51B depict schematics of cicuits for the production of indole metabolites. FIG. 51A depicts a schematic of an indole-3-propionic acid (IP A) synthesis circuit. IPA produced by the gut micro bioata has a significant positive effect on barrier integrity. IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). In some embodiments, IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole- 3 -aery late reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3- acrylic acid, and indole- 3 -aery late reductase converts indole- 3 -acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. The strains further comprise optional circuits for tryptophan production are as depicted and described in FIG. 41.

[0077] FIG. 51B depicts a schematic of another indole-3-propionic acid (IPA) synthesis circuit. Enzymes are as follows: 1. TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldHl/FldH2: indole- 3 -lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole- 3 -lactate dehydratase, e.g., from

Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; Acul: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate, NH ₃ , NAD(P)H and H ⁺ . Indole- 3 -lactate dehydrogenase ((EC 1.1.1.110, e.g. , Clostridium sporogenes or Lactobacillus casei) converts (indol-3yl)pyruvate and NADH and H+ to indole-3-lactate and NAD+. Indole- 3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) converts indole- 3 -lactate and indol- 3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (FldD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3- propionyl-CoA. Indole- 3 -lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole- 3-acrylyl-CoA. The strains further comprise optional circuits for tryptophan production are as depicted and described in FIG. 41.

[0078] FIG. 52A, FIG. 52B, FIG. 52C, FIG. 52D, and FIG. 52E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g. , FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g. , deletion of thyA (Δ thyA; thymidine dependence). FIG. 52A depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 41 and/or described in the description of FIG. 41 and/or FIG. 52B. Optionally, Trp Repressor and/or the tnaA gene (encoding a tryptophanase converting Trp into indole) are deleted to further increase levels of tryptophan produced. The bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan. FIG. 52B depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further comprises either a wild type or a feedback resistant SerA gene. Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD 1 to NADH. E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 41 and/or described in the description of FIG. 41. Optionally, Trp Repressor and/or the tnaA gene (encoding a tryptophanase converting Trp into indole) are deleted to further increase levels of tryptophan produced. The bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan. FIG. 52C depicts non-limiting example of a tryptamine producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises additional circuits as depicted and/or described in FIG. 52A and/or FIG. 52B and/or FIG. 41. Additionally, the strain comprises tdc

(tryptophan decarboxylase, e.g., from Catharanthus roseus), which converts tryptophan into tryptamine. FIG. 52D depicts a non-limiting example of an indole- 3 -acetate producing strain. Tryptophan optionally is produced from chorismate precursor, and the strain optionally comprises additional circuits as depicted and/or described in FIG. 52A and/or FIG. 52B and/or FIG. 41. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from

Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole- 3 -acetaldehyde into indole- 3 -acetate. FIG. 52E depicts a non-limiting example of an indole-3-propionate-producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises additional circuits as depicted and/or described in FIG. 52A and/or FIG. 52B and/or FIG. 41.

[0079] FIG. 53 depicts a schematic of the trypophan catabolic pathway/indole biosynthesis pathways. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al, CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016)). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 53. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 53, including but not limited to, kynurenine, indole-3- aldehyde, indole- 3 -acetic acid, and/or indole-3 acetaldehyde. Description of Embodiments

[0080] The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating or treating disorders or conditions that are caused by toxic molecules, metabolites, or other deleterious molecules, e.g. , chemotherapy- induced diarrhea. The genetically engineered bacteria are capable of reducing and/or metabolizing the toxic molecule, metabolite, or deleterious molecule, particularly in low- oxygen conditions, such as in the mammalian gut. In some embodiments, the engineered bacteria are further capable of producing one or more anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the engineered bacteria are further capable of producing one or more anti- inflammation and/or gut barrier function enhancer molecule(s) in inducing environments, e.g., in the gut.

[0081] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0082] As used herein, the term "toxin" in the context of a kill switch being expressed in a recombinant bacterium of the disclosure refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure. The term "toxin" in the context of a kill switch being expressed in a recombinant bacterium of the disclosure is intended to include bacteriostatic proteins and bactericidal proteins. The term "toxin" is intended to include, but not limited to, lytic proteins, bacteriocins (e.g. , microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term "anti-toxin" or "antitoxin," as used herein in the context of a kill switch being expressed in a recombinant bacterium of the disclosure , refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term anti-toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins in the context of a kill switch being expressed in a recombinant bacterium of the disclosure are known in the art and described in more detail infra.

[0083] As used herein, the term "toxic molecule" and its cognates (meant to be distinguished from the term "toxin" used in the context of kill- switch) aare used to refer to a substance that is deleterious to the health of an organism, e.g., a human subject. Non- limiting examples of such toxic molecules include heavy metals, chemotherapeutic drugs, nonsteroidal ant i- inflammatory drugs (NSAIDs), natural toxic molecules such as botulinum and tetanus toxic molecules, and synthetic toxic molecules such as dioxin and sarin. In some instances, a toxic molecule may be a substance that has predominantly deleterious effects, e.g., lead poisoning and sarin poisoning. In other instances, a toxic molecule may be a substance that has some therapeutically beneficial effects, e.g., chemotherapeutic drugs and NSAIDs. In some instances, a toxic molecule has therapeutically beneficial effects and its metabolites or byproducts have deleterious effects; for example, irinotecan is an effective antineoplastic agent, and its metabolite SN-38 causes gastrointestinal toxicity and dose-limiting diarrhea (Stein et al., 2010). In other instances, a toxic molecule has therapeutically beneficial effects as well as deleterious effects; for example, methotrexate is an effective antineoplastic agent, but can cause bone marrow and gastrointestinal toxicity (Chabner et al, 1972). In some embodiments, a toxic molecule is therapeutically beneficial or innocuous at particular doses, e.g., low doses, but toxic at other doses, e.g., high doses.

[0084] Non-limiting examples of toxic chemotherapeutic drugs include antimetabolites, methotrexate, gemcitabine, cytosine arabinoside, fluoropyrimidines, fluorouracil, capecitabine, tegafur-uracil, multitargeted folinic acid antagonists, pemetrexed, raltitrexed, gemcitabine, plant alkaloids, vinca alkaloids, vincristine, vinorelbine, epipodophyllotoxins, etoposide, taxanes, paclitaxel, docetaxel, topoisomerase I inhibitors, irinotecan, cytotoxic antibiotics, anthracyclines, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, alkylating agents, cyclophosphamide, platinums, cisplatin, carboplatin, oxaliplatin, nedaplatin, antibodies, ipilumumab, antibodies against VEGF, bevacizumab, tyrosine-kinase inhibitors, EGFR inhibitors, lapatinib, cetuximab, and their metabolites or byproducts, e.g., SN-38 (Andreyev et al., 2014). These toxic molecules are capable of causing clinically important diarrhea that can dose-limit or delay the chemotherapeutic drug and/or interfere with or detract from cancer treatment, which can impact survival (Stein et al., 2010).

[0085] Non-limiting examples of toxic NSAIDs include etoricoxib, etodolac, rofecoxib, meloxicam, celecoxib, piroxicam, naproxen, indomethacin, ketoprofen, piroxicam, ibuprofen, diclofenac, and COX-2 inhibitors. These toxic molecules are capable of causing clinically important diarrhea that can dose-limit, delay, and/or interfere with or detract from treatment.

[0086] Non-limiting examples of toxic heavy metals include aluminum, antimony, arsenic, barium, bismuth, cadmium, chromium, cobalt, copper, gold, iron, lead, lithium, manganese, mercury, nickel, phosphorous, platinum, selenium, silver, thallium, tin, and zinc. These toxic molecules are capable of causing heavy metal poisoning.

[0087] "Deleterious molecules" is used to refer to the toxic molecules, heavy metals, chemotherapeutic drugs, NSAIDs, and their metabolites and byproducts as described above, as well as other compounds that adversely affect health, e.g. , radioactive substances and pesticides. As used herein, a "deleterious" molecule may have therapeutically beneficial effects, e.g. , antineoplastic/cytotoxic effects on cancerous cells.

[0088] "Detoxify" is used to refer to the process or processes, natural or synthetic, by which the above-referenced deleterious molecules are removed, inhibited, metabolized, and/or converted into one or more non-toxic molecules. The deleterious molecule may be detoxified directly or indirectly. For example, detoxification may refer to the inhibition of a toxic substance, e.g. , a small molecule that inhibits β-glucuronidase to prevent the conversion of irinotecan into the toxic metabolite SN-38 (Wallace et al., 2010). Alternatively, detoxification may refer to the conversion of a toxic substance into a non-toxic substance, e.g., an enzyme that metabolizes SN-38 into one or more non-toxic molecules, or an enzyme that reattaches glucuronide to SN-38, thereby converting it into non-toxic SN-38G. In some embodiments, detoxification refers to the modification of the intestinal microflora such that toxic molecule- promoting bacteria are reduced, e.g. , commensal bacteria that convert SN-38G into toxic SN- 38. In some embodiments, the toxic molecule, e.g., a chemotherapeutic drug or NSAID, is capable of exerting both therapeutically beneficial effects and deleterious effects, and the genetically engineered bacteria detoxify the deleterious molecule after the therapeutically beneficial effects have been exerted.

[0089] As used herein, "payload" refers to one or more molecules of interest to be produced by a genetically engineered bacterium. In some embodiments, the payload is a therapeutic payload, which refers to a molecule, substance, or drug that is useful for modulating or treating a disorder or condition, e.g., chemotherapy- induced diarrhea. For example, the condition is methotrexate-induced diarrhea, and the payload is carboxypeptidase Gi (CPD Gi) (Chabner et al., 1972) or CPD G ₂ . In some embodiments, the payload is a therapeutic molecule encoded by a gene. In alternate embodiments, the payload is a therapeutic molecule produced by a biosynthetic pathway, e.g. , butyrate (Vidyasagar et al., 2002). In some embodiments, the genetically engineered bacterium comprises two or more payloads; for example, the condition is methotrexate-induced diarrhea, and the first payload is carboxypeptidase G ₂ encoded by a CPD G ₂ gene, and the second payload is butyrate produced by a gene cassette encoding the genes of the butyrate biosynthesis pathway. [0090] In some embodiments, the payload is a therapeutic payload capable of detoxifying a toxic or deletorious molecule. These therapeutic or payload molecules are also referred to herein as "detoxification molecules". Exemplary detoxifcation molecules include, but are not limited to, carboxypeptidase Gi (CPD Gi) or carboxypeptidase G ₂ (CPD G ₂ ), D- saccharic acid 1, 4-lactone (SAL), a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, a proton pump inhibitor (e.g. , omeprazole, aspirin, lansoprazole,

dexlansoprazole, rabeprazole, pantoprazole, esomeprazole, esomeproazole

magnesium/naproxen, and omeprazole/sodium bicarbonate), a heavy metal chelator, a plant phytochelatin. In some embodiments, the payload is an antiinflammatory or gut barrier enhancer molecule, e.g. butyrate, acetate, propionate, GLP-2, IL- 10, IL-22, IL-2, other interleukins, and/or tryptophan and/or one or more of its metabolites. In some embodiments, the payload is a regulatory molecule, e.g. , a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments the payload comprises an antibiotic resistance gene or genes. In some embodiments, the payload is encoded by a gene, multiple genes, gene cassette, or an operon. In alternate embodiments, the payload is produced by a bio synthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads.

[0091] As used herein, "diseases and conditions associated with gut inflammation and/or compromised gut barrier function" include, but are not limited to, inflammatory bowel diseases, diarrheal diseases, and related diseases. "Inflammatory bowel diseases" and "IBD" are used interchangeably herein to refer to a group of diseases associated with gut

inflammation, which include, but are not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, and indeterminate colitis. As used herein, "diarrheal diseases" include, but are not limited to, acute watery diarrhea, e.g., cholera; acute bloody diarrhea, e.g. , dysentery; and persistent diarrhea. As used herein, related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis.

[0092] Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of diarrhea, bloody stool, mouth sores, perianal disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron deficiency, anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal development, inflammation of the skin, inflammation of the eyes, inflammation of the joints, inflammation of the liver, and inflammation of the bile ducts. These symptoms relating to the disruption of gut health and other symptoms relating to an inflammatory state may arise as a result of a subject's exposure to one or more toxic or deleterious molecules.

[0093] As used herein, grade 1 diarrhea, grade 2 diarrhea, grade 3 diarrhea, grade 4 diarrhea, and grade 5 diarrhea are all encompassed by the term "diarrhea." In general, grade 1 diarrhea is characterized by an increase of < 4 bowel movements per day or a mild increase in stoma output. Grade 2 diarrhea is characterized by an increase of 4-6 bowel movements per day, a moderate increase in stoma output, and moderate cramping or nocturnal stools. Grade 3 diarrhea is characterized by an increase of 7-9 bowel movements per day, incontinence, or severe increase in stoma output, and severe cramping or nocturnal stools. Grade 4 diarrhea is generally life-threatening and is characterized by an increase of more than 10 bowel movements per day, grossly bloody stool, severe dehydration, extremely low blood pressure, and/or need for parenteral support. Grade 5 diarrhea generally results in death. See, e.g., Andreyev et al., 2014; National Cancer Institute Common Toxicity Criteria for Diarrhea.

Diarrhea may optionally be co-morbid with steatorrhea (excess fat in the stool due to fat malabsorption in the gut).

[0094] As used herein, "anti-inflammation molecules" and/or "gut barrier function enhancer molecules" include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP- 1, IL- 10, IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD ₂ , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein. Such molecules may also include compounds that inhibit pro-inflammatory molecules, e.g. , a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-γ, IL- Ιβ, IL-6, IL-8, IL- 17, and/or chemokines, e.g. , CXCL-8 and CCL2. Such molecules also include AHR agonists {e.g. , which result in IL-22 production, e.g. , indole acetic acid, indole-3-aldehyde, and indole) and and PXR agonists {e.g. , IP A), as described herein. Such molecules also include HDAC inhibitors (e.g., butyrate), activators of GPR41 and/or GPR43 (e.g., butyrate and/or propionate and/or acetate), activtators of

GPR109A (e.g. , butyrate), inhibitors of NF-kappaB signaling (e.g. , butyrate), and modulators of PPARgamma (e.g. , butyrate), activators of AMPK signaling (e.g. , acetate), and modulators of GLP- 1 secretion. Such molecules also include hydro xyl radical scavengers and antioxidants (e.g., IP A). A molecule may be primarily ant i- inflammatory, e.g., IL- 10, or primarily gut barrier function enhancing, e.g. , GLP-2. A molecule may be both anti- inflammatory and gut barrier function enhancing. An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g. , elafin is encoded by the PI3 gene.

Alternatively, an anti- inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g. , butyrate. These molecules may also be referred to as therapeutic molecules. In some instances, the "anti- inflammation molecules" and/or "gut barrier function enhancer molecules" are referred to herein as "effector molecules" or "therapeutic molecules" or "therapeutic polypeptides".

[0095] A disease or condition associated with gut inflammation and/or compromised gut barrier function may be an autoimmune disorder. A disease or condition associated with gut inflammation and/or compromised gut barrier function may be co-morbid with an autoimmune disorder. As used herein, "autoimmune disorders" include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic

leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospho lipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osto myelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn' s disease, Cogan' s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus,

Dressier' s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis.

[0096] As used herein, the term "polypeptide" includes "polypeptide" as well as "polypeptides," and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, "peptides," "dipeptides," "tripeptides, "oligopeptides," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post- expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term "peptide" or "polypeptide" may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.

[0097] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required.

Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms "fragment," "variant," "derivative" and "analog" include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non-naturally occurring. No n- naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0098] As used herein, the term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones.

[0099] Polypeptides also include fusion proteins. As used herein, the term "variant" includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term "fusion protein" refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion

proteins. "Derivatives" include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution.

Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C.

(1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, He, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

[00100] An antibody generally refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen. An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond. The recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively.

[00101] As used herein, the term "antibody" or "antibodies"is meant to encompasses all variations of antibody and fragments thereof that possess one or more particular binding specificities. Thus, the term "antibody" or "antibodies" is meant to include full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single domain antibodies (sdAB, VHH framents), heavy chain antibodies (HCAb), nanobodies, diabodies, and minibodies. Antibodies can have more than one binding specificity, e.g. , be bispecific. The term "antibody" is also meant to include so-called antibody mimetics. Antibody mimetics refers to small molecules, e.g. , 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure. Antibody mimetics, include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B crystalline), Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from Sulfolobus acidocaldarius), Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif),

Fynomers (SH3 domain of Fyn), Kunitz domain peptides Kunitz domains of various protease inhibitors), Ecallantide (Kalbitor), and Monobodies. In certain aspects, the term "antibody" or "antibodies" is meant to refer to a single chain antibody(ies), single domain antibody(ies), and camelid antibody(ies). Utility of antibodies in the treatment of cancer and additional anti cancer antibodies can for example be found in Scott et al, Antibody Therapy for Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference in its entirety.

[00102] A "single-chain antibody" or "single-chain antibodies" typically refers to a peptide comprising a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and optionally a linker or bond, such as a disulfide bond. The single-chain antibody lacks the constant Fc region found in traditional antibodies. In some embodiments, the single-chain antibody is a naturally occurring single-chain antibody, e.g., a camelid antibody. In some embodiments, the single-chain antibody is a synthetic, engineered, or modified single-chain antibody. In some embodiments, the single-chain antibody is capable of retaining substantially the same antigen specificity as compared to the original immunoglobulin despite the addition of a linker and the removal of the constant regions. In some aspects, the single chain antibody can be a "scFv antibody", which refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins (without any constant regions), optionally connected with a short linker peptide of ten to about 25 amino acids, as described, for example, in U.S. Patent No. 4,946,778, the contents of which is herein incorporated by reference in its entirety. The Fv fragment is the smallest fragment that holds a binding site of an antibody, which binding site may, in some aspects, maintain the specificity of the original antibody. Techniques for the production of single chain antibodies are described in U.S. Patent No.

4,946,778. The Vh and VL sequences of the scFv can be connected via the N-terminus of the VH connecting to the C-terminus of the VL or via the C-terminus of the VH connecting to the N-terminus of the VL. ScFv fragments are independent folding entities that can be fused indistinctively on either end to other epitope tags or protein domains. Linkers of varying length can be used to link the Vh and VL sequences, which the linkers can be glycine rich (provides flexibility) and serine or threonine rich (increases solubility). Short linkers may prevent association of the two domains and can result in multimers (diabodies, tribodies, etc.). Long linkers may result in proteolysis or weak domain association (described in Voelkel et al el., 2011). Linkers of length between 15 and 20 amino acids or 18 and 20 amino acids are most often used. Additional non- limiting examples of linkers, including other flexible linkers are described in Chen et al, 2013 (Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Fusion Protein Linkers: Property, Design and Functionality), the contents of which is herein incorporated by reference in its entirety. Flexible linkers are also rich in small or polar amino acids such as Glycine and Serine, but can contain additional amino acids such as Threonine and Alanine to maintain flexibility, as well as polar amino acids such as Lysine and Glutamate to improve solubility. Exemplary linkers include, but are not limited to, (Gly-Gly-Gly-Gly-Ser)n, KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8, and Gly and Ser rich flexible linker, GSAGSAAGSGEF. "Single chain antibodies" as used herein also include single- domain antibodies, which include camelid antibodies and other heavy chain antibodies, light chain antibodies, including nanobodies and single domains VH or VL domains derived from human, mouse or other species. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies include domain antigen-binding units which have a camelid scaffold, derived from camels, llamas, or alpacas. Camelids produce functional antibodies devoid of light chains. The heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen-binding molecules (Fabs) or single chain variable fragments (scFvs). Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies. Camelid scaffold-based antibodies can be produced using methods well known in the art. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. Alternatively, the dimeric variable domains from IgG from humans or mice can be split into monomers. Nanobodies are single chain antibodies derived from light chains. The term "single chain antibody" also refers to antibody mimetic s.

[00103] As used herein the term "linker", "linker peptide" or "peptide linkers" or

"linker" refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g. , that link two polypeptide domains. As used herein the term "synthetic" refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.

[00104] In some embodiments, the antibodies expressed by the engineered microorganisms are bispecfic. In certain embodiments, a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope. Antigen-binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Monomeric single-chain diabodies (scDb) are readily assembled in bacterial and mammalian cells and show improved stability under physiological conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001) 14 (10): 815-823 (describes optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies).

[00105] As used herein, the term "gene" refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. In one embodiment, a "gene" does not include regulatory sequences preceding and following the coding sequence. A "native gene" refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A "chimeric gene" refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature.

[00106] As used herein, the term "gene sequence" is meant to refer to a genetic sequence, e.g. , a nucleic acid sequence. The gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g. , a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence.

[00107] In some embodiments, the term "gene" or "gene sequence" is meant to refer to a nucleic acid sequence encoding a payload capable of detoxifying a deleterious molecule and/or any of the anti- inflammatory and gut barrier function enhancing molecules described herein, e.g. , IL-2, IL-22, superoxide dismutase (SOD), kynurenine, GLP-2, GLP- 1, IL- 10, IL-27, TGF-βΙ, TGF-β2, N-acylphosphatidylethanolamines (NAPEs), elafin, and trefoil factor, as well as others. The nucleic acid sequence may comprise the entire gene sequence or a partial gene sequence encoding a functional molecule. The nucleic acid sequence may be a natural sequence or a synthetic sequence. The nucleic acid sequence may comprise a native or wild-type sequence or may comprise a modified sequence having one or more insertions, deletions, substitutions, or other modifications, for example, the nucleic acid sequence may be codon-optimized.

[00108] As used herein, a "heterologous" gene or "heterologous sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell and can be a native sequence (naturally found or expressed in the cell) or non- native sequence (not naturally found or expressed in the cell) and can be a natural or wild-type sequence or a variant, non-natural, or synthetic sequence. "Heterologous gene" includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, the term "transgene" refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.

[00109] As used herein, a "gene cassette" or "operon" encoding a biosynthetic pathway refers to the two or more genes that are required to produce a molecule of interest, e.g. , a gut barrier function enhancer molecule such as butyrate. In addition to encoding a set of genes capable of producing the molecule of interest, the gene cassette may also comprise additional transcription and translation elements, e.g. , a ribosome binding site.

[00110] A "butyrogenic gene cassette," "butyrate biosynthesis gene cassette," and "butyrate operon" are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. The genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria. A butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C- acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate

butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013). One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g. , codon optimized. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiAl from

Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from

Treponema denticola. The butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.

[00111] Likewise, a "propionate gene cassette" or "propionate operon" refers to a set of genes capable of producing propionate in a biosynthetic pathway. Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. The genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria. In some embodiments, the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and

Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyses the reduction of lactate to propionate. Dehydration of (K)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA by lactoyl-CoA dehydratase (LcdABC). Acrolyl-CoA is converted to propionyl-CoA by acrolyl-CoA reductase (EtfA, AcrBC). In some embodiments, the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the acul gene from R. sphaeroides. This gene product catalyzes the NADPH-dependent acrylyl- CoA reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides; Asao 2013). Thus the propionate cassette comprises pet, IcdA, IcdB, IcdC, and acul. In another embodiment, the homo log of Acul in E coli, YhdH is used (see. e.g., Structure of Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004). This the propionate cassette comprises pet, IcdA, IcdB, IcdC, and yhdH. In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.

[00112] In another example of a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering Escherichia coli for high-level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g. by the genetically engineered bacteria of the disclosure. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm (methylmalonyl-CoA mutase) converts succinyl CoA to L-methylmalonylCoA, YgfD is a Sbm- interacting protein kinase with GTPase activity, ygfG (methylmalonylCoA decarboxylase) converts L-methylmalonylCoA into PropionylCoA, and ygfH (propionyl-CoA/succinylCoA transferase) converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009). This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI: 18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, beep) which converts methylmalonyl-CoA to propionyl-CoA.

[00113] The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate. One or more of the propionate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

[00114] An "acetate gene cassette" or "acetate operon" refers to a set of genes capable of producing acetate in a biosynthetic pathway. Bacteria "synthesize acetate from a number of carbon and energy sources," including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008). The genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria. Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994). Several bacteria, such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or C0 ₂ + H ₂ into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art. The acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

[00115] Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein. In some

embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions. Thus, for example, each gene or gene cassette for producing a payload may be present on a plasmid or bacterial chromosome.

[00116] As used herein the term "codon-optimized" refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A "codon-optimized sequence" refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[00117] "Reduced" levels of a toxic molecule, metabolite, or other deleterious molecule is used to refer to a reduction in the amount of said toxic molecule, metabolite, or molecule after treatment with the genetically engineered bacteria of the invention, as compared to amount after treatment with unmodified bacteria of the same subtype under the same conditions. In some embodiments, reduction is measured by comparing the level of the toxic molecule, metabolite, or other deleterious molecule before and after administering the genetically engineered bacteria of the invention. In some embodiments, levels of the toxic molecule, metabolite, or other deleterious molecule is about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% as compared to levels in an untreated or control condition.

Each gene or gene cassette may be operably linked to a promoter that is induced by exogenous environmental conditions, for example low-oxygen conditions, as is found in a mammalian gut. "Operably linked" refers a nucleic acid sequence, e.g., a carboxypeptidase G ₂ (CPD G ₂ ) gene, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

A regulatory region "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding an anti- inflammatory or gut barrier enhancer molecule, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the anti- inflammatory or gut barrier enhancer molecule. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be "directly linked" to a regulatory sequence in a manner which allows expression of the gene. In another embodiment, a gene may be "indirectly linked" to a regulatory sequence in a manner which allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes.

[00118] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR- responsive promoters, and DNR-responsive promoters are known in the art {see, e.g.,

Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1.

[00119] In a non-limiting example, a promoter (PfnrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic conditions by the global

transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS. Table 1. Examples of transcription factors and responsive genes and regulatory regions

[00120] As used herein, a "tunable regulatory region" refers to a nucleic acid sequence under direct or indirect control of a transcription factor and which is capable of activating, repressing, derepressing, or otherwise controlling gene expression relative to levels of an inducer. In some embodiments, the tunable regulatory region comprises a promoter sequence. The inducer may be RNS, or other inducer described herein, and the tunable regulatory region may be a RNS -responsive regulatory region or other responsive regulatory region described herein. The tunable regulatory region may be operatively linked to a gene sequence(s) or gene cassette for the production of one or more payloads, e.g. , a butyrogenic or other gene cassette or gene sequence(s). For example, in one specific embodiment, the tunable regulatory region is a RNS-derepressible regulatory region, and when RNS is present, a RNS- sensing transcription factor no longer binds to and/or represses the regulatory region, thereby permitting expression of the operatively linked gene or gene cassette. In this instance, the tunable regulatory region derepresses gene or gene cassette expression relative to RNS levels. Each gene or gene cassette may be operatively linked to a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one RNS.

[00121] In some embodiments, the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS). In some embodiments, exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut. In some embodiments, the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules.

[00122] In some embodiments, the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in

combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response). In some embodiments, the loss of exposure to an exogenous environmental condition (for example, in vivo) inhibits the activity of an inducible promoter, as the exogenous environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut). "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the

gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.

[00123] As used herein, a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non- naturally occurring sequence (see, e.g., Purcell et al., 2013). The non- native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, "non-native" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a payload that is operably linked to a promoter that is not associated with said gene or gene cassette in nature, e.g. , a FNR-responsive promoter operably linked to a butyrogenic gene cassette. In some embodiments, the genetically engineered microorganism of the disclosure comprises a gene that is operably linked to a promoter that is not associated with said gene in nature. For example, in some embodiments, the genetically engineered bacteria disclosed herein comprise a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., an FNR responsive promoter (or other promoter disclosed herein) operably linked to an ant i- inflammatory or gut barrier enhancer molecule. In some embodiments, the genetically engineered virus of the disclosure comprises a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., a promoter operably linked to a gene encoding an antiinflammatory or gut barrier enhancer molecule.

[00124] As used herein, the term "coding region" refers to a nucleotide sequence that codes for a specific amino acid sequence. The term "regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, signal sequences, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter or other promoter disclosed herein.

[00125] A "promoter" as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5' of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g. , in a cell- or tissue- specific manner, in response to different

environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. A "constitutive promoter" refers to a promoter that allows for continual transcription of the coding sequence or gene under its control.

[00126] "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli σ promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of

Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli σ 32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ 70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110

(BBa_M13110)), a constitutive Bacillus subtilis σ ^Α promoter (e.g., promoter veg

(BBa_K143013), promoter 43 (BBa_K143013), P _liaG (BBa_K823000), P _lepA (BBa_K823002), P _veg (BBa_K823003)), a constitutive Bacillus subtilis σ promoter (e.g., promoter etc

(BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997;

BBa_K113010; BBa_Kl 13011 ; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181 ;

BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)).

[00127] An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. An "inducible promoter" refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A "directly inducible promoter" refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An "indirectly inducible promoter" refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by "inducible promoter." Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g. , arabinose and

tetracycline. Examples of inducible promoters include, but are not limited to, an FNR responsive promoter, a ParaC promoter, a ParaBAD promoter, and a PTetR promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.

[00128] As used herein, genetically engineered bacteria that "overproduce" a payload refer to bacteria that produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50- fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400- fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800- fold, at least about 900- fold, at least about 1,000- fold, or at least about 1,500- fold more payload than unmodified bacteria of the same subtype under the same conditions. In some

embodiments, the mRNA transcript levels of one or more of the gene(s) for producing the payload in the genetically engineered bacteria are at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800- fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold higher than the mRNA transcript levels in unmodified bacteria of the same subtype under the same conditions. In certain embodiments, the unmodified bacteria will not have detectable levels of the payload and/or transcription of the gene(s). However, protein and/or transcription levels of the payload will be detectable in the corresponding genetically engineered bacterium. Transcription levels may be detected by directly measuring mRNA levels of the genes.

Methods of measuring of the payload and/or transcript are known in the art. Levels of lead in the blood, for example, may be measured by atomic absorption spectrometry, anodic stripping voltammetry, and/or mass spectrometry. Level of methotrexate in the blood, for example, may be measured by HPLC, immunoassay, and/or enzyme inhibition assay. [00129] "Exogenous environmental condition(s)" or "environmental conditions" refer to settings or circumstances under which the promoter described herein is directly or indirectly induced. The phrase is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject environment. Thus, "exogenous" and "endogenous" may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous

environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease- state, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue-specific or disease- specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics.

[00130] As used herein, "exogenous environmental conditions" or

"environmental conditions" refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced. The phrase

"exogenous environmental conditions" is meant to refer to settings or circumstances or environmental conditions external to the engineered microorganism, which relate to in vitro culture conditions of the microorganism. "Exogenous environmental conditions" may also refer to the conditions during growth, production, and manufacture of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low oxygen culture conditions and other conditions under set oxygen concentrations. Such conditions also include the presence of a chemical and/or nutritional inducer, such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium. Such conditions also include the temperatures at which the microorganisms are grown prior to in vivo administration. For example, using certain promoter systems, certain temperatures are permissive to expression of a payload, while other temperatures are non-permissive. Oxygen levels, temperature and media composition influence such exogenous environmental conditions. Such conditions affect proliferation rate, rate of induction of the payload, and overall viability and metabolic activity of the strain during strain production.

[00131] As used herein, "exogenous" and "endogenous" may be used

interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue- specific or disease- specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is specific to an inflammatory disease. In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An "oxygen level-dependent promoter" or "oxygen level-dependent regulatory region" refers to a nucleic acid sequence to which one or more oxygen level- sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.

[00132] As used herein, the term "low oxygen" is meant to refer to a level, amount, or concentration of oxygen (0 ₂ ) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., <21% 0 _2; <160 torr 0¾). Thus, the term "low oxygen condition or conditions" or "low oxygen environment" refers to conditions or environments containing lo wer levels of o ygen than are present in the atmosphere. In some embodiments, the term "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0 ₂ ) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term "low oxygen" is meant to refer to a level, amount, or concentration of 0 ₂ that is 0-60 mmHg 0 ₂ (0-60 torr 0 ₂₎ (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg 0 ₂ ), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg 0 ₂ , 0.75 mmHg 0 ₂ , 1.25 mmHg 0 ₂ , 2.175 mmHg 0 ₂ , 3.45 mmHg 0 ₂ , 3.75 mmHg 0 ₂ , 4.5 mmHg 0 ₂ , 6.8 mmHg 0 ₂ , 11.35 mmHg 02, 46.3 mmHg 0 ₂ , 58.75 mmHg, etc., which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way). In some embodiments, "low oxygen" refers to about 60 mmHg 0 ₂ or less (e.g., 0 to about 60 mmHg 0 ₂ ). The term "low oxygen" may also refer to a range of 0 ₂ levels, amounts, or concentrations between 0-60 mmHg 0 ₂ (inclusive), e.g., 0-5 mmHg 0 ₂ , < 1.5 mmHg 0 ₂ , 6-10 mmHg, < 8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way. See, for example, Albenberg et al,

Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al, J Clin. Invest., 41(11): 1971- 1980 (1962); Crompton et al, J Exp. Biol., 43: 473-478 (1965); He et al, PNAS (USA), 96: 4586-4591 (1999); McKeown, Br. J. Radiol., 87:20130676 (2014) (doi: 10.1259/brj.20130676), each of which discusses the oxygen levels found in the mammalian gut of various species and each of which are incorportated by reference herewith in their entireties. In some embodiments, the term "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0 ₂ ) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments, "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0 ₂ ) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions. For example, Table A summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (0 ₂ ) is expressed as the amount of dissolved oxygen ("DO") which refers to the level of free, non-compound oxygen (0 ₂ ) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; lmg/L = 1 ppm), or in micromoles (umole) (1 umole 0 ₂ = 0.022391 mg/L 0 ₂ ). Fondriest Environmental, Inc., "Dissolved Oxygen",

Fundamentals of Environmental Measurements, 19 Nov 2013,

www. fondriest. com/environmental- measurements/parameters/water- quality/dissolved- oxygen/>. In some embodiments, the term "low oxygen" is meant to refer to a level, amount, or concentration of oxygen (0 ₂ ) that is about 6.0 mg/L DO or less, e.g. , 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g. , 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way. The level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (0 ₂ ) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium). Weil-aerated solutions (e.g., solutions subjected to mixing and/or stirring) without oxygen producers or consumers are 100% air saturated. In some embodiments, the term "low oxygen" is meant to refer to 40% air saturation or less, e.g. , 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-40%, inclusive (e.g. , 0-5%, 0.05 - 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0- 10%, 5- 10%, 10- 15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. In some embodiments, the term "low oxygen" is meant to refer to 9% 0 ₂ saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, 0 ₂ saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of 0 ₂ saturation levels between 0-9%, inclusive {e.g. , 0-5%, 0.05 - 0.1%, 0.1-0.2%, 0.1- 0.5%, 0.5 - 2.0%, 0-8%, 5-7%, 0.3-4.2% 0 _2, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.

Table A.

[00133] "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g. , in the gastrointestinal tract, and particularly in the intestines.

[00134] "Microorganism" refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, e.g. ,

Saccharomyces, and protozoa. In some aspects, the microorganism is engineered ("engineered microorganism") to produce one or more therpauetic molecules, e.g. , an antinflammatory or barrier enhancer molecule. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus. [00135] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides,

Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and

Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.

[00136] "Probiotic" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei,

Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

[00137] As used herein, "stably maintained" or "stable" bacterium is used to refer to a bacterial host cell carrying non- native genetic material, e.g., a gene or gene cassette for producing a payload, which is incorporated into the host genome or propagated on a self- replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium is capable of survival and/or growth in vitro (e.g., in medium) and/or in vivo (e.g., in the gut). For example, the stable bacterium may be a genetically engineered bacterium comprising a butyrogenic gene cassette, in which the plasmid or chromosome carrying the gene cassette is stably maintained in the bacterium, such that the gene cassette can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, the stable bacterium may be a genetically engineered bacterium comprising a gene encoding a encoding a payload, e.g., one or more anti- inflammation and/or gut barrier enhancer molecule(s), in which the plasmid or chromosome carrying the gene is stably maintained in the bacterium, such that the payload can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.

[00138] As used herein, the term "recombinant microorganism" refers to a microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state. Thus, a "recombinant bacterial cell" or "recombinant bacteria" refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

[00139] A "programmed or engineered microorganism" refers to a

microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state to perform a specific function. Thus, a "programmed or engineered bacterial cell" or "programmed or engineered bacteria" refers to a bacterial cell or bacteria that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.

[00140] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.

[00141] As used herein, the term "plasmid" or "vector" refers to an

extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell' s genome. Plasmids are usually circular and capable of autonomous replication. Plasmids may be low-copy, medium-copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g. , a gene encoding a detoxification molecule, an ant i- inflammatory molecule, or a gut barrier enhancer molecule.

[00142] As used herein, the term "transform" or "transformation" refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically- stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.

[00143] The term "genetic modification," as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, base substitution, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter

modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g. , a plasmid comprising gene sequene(s) encoding a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g. , introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation.

[00144] As used herein, the term "genetic mutation" refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example, substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g. , one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term "genetic mutation" is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g. , enzymatic activity) of the gene's polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.

[00145] As used herein, the term "transporter" is meant to refer to a mechanism, e.g. , protein, proteins, or protein complex, for importing a molecule, e.g., amino acid, peptide (di-peptide, tri-peptide, polypeptide, etc), toxic molecule, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.

[00146] As used herein, the term "modulate" and its cognates means to alter, regulate, or adjust positively or negatively a molecular or physiological readout, outcome, or process, to effect a change in said readout, outcome, or process as compared to a normal, average, wild-type, or baseline measurement. Thus, for example, "modulate" or "modulation" includes up-regulation and down-regulation. A non- limiting example of modulating a readout, outcome, or process is effecting a change or alteration in the normal or baseline functioning, activity, expression, or secretion of a biomolecule (e.g. a protein, enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid, or other compound). Another non- limiting example of modulating a readout, outcome, or process is effecting a change in the amount or level of a biomolecule of interest, e.g. in the serum and/or the gut lumen. In another non- limiting example, modulating a readout, outcome, or process relates to a phenotypic change or alteration in one or more disease symptoms. Thus, "modulate" is used to refer to an increase, decrease, masking, altering, overriding or restoring the normal functioning, activity, or levels of a readout, outcome or process (e.g, biomolecule of interest, and/or molecular or physiological process, and/or a phenotypic change in one or more disease symptoms).

[00147] As used herein, the terms "modulate" and "treat" and their cognates refer to an amelioration of a disorder or condition, e.g. , heavy metal poisoning, or at least one discernible symptom thereof. In another embodiment, "modulate" and "treat" refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "modulate" and "treat" refer to inhibiting the progression of a disorder or condition, either physically (e.g. , stabilization of a discernible symptom), physiologically (e.g. , stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to slowing the progression or reversing the progression of a disorder or condition. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disorder or condition.

[00148] Those in need of treatment may include individuals already having a particular disorder or condition, as well as those at risk of having, or who may ultimately acquire the disorder or condition, e.g. , chemotherapy- induced diarrhea. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treatment may encompass reducing or eliminating one or more deleterious symptoms, e.g. , diarrhea, and does not necessarily encompass the elimination of the underlying disorder.

[00149] Treating the diseases described herein may encompass increasing levels of butyrate, increasing levels of acetate, increasing levels of butyrate and increasing GLP-2, IL- 22, and/o rIL- 10, and/or modulating levels of tryptophan and/or its metabolites (e.g. , kynurenine), and/or providing any other detoxification of a deleterious molecule and/or gut barrier enhancer molecule and does not necessarily encompass the elimination of the underlying disease.

[00150] As used herein a "pharmaceutical composition" refers to a preparation of genetically engineered bacteria of the invention with other components such as a

physiologically suitable carrier and/or excipient.

[00151] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases. [00152] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

Examples include, but are not limited to, calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[00153] The terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g., chemotherapy- induced diarrhea. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder or condition caused by a toxic molecule, metabolite, or other deleterious molecule. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

[00154] As used herein, the term "bacteriostatic" or "cytostatic" refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of recombinant bacterial cell of the disclosure.

[00155] As used herein, the term "bactericidal" refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.

[00156] As used herein, the terms "secretion system" or "secretion protein" refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g. , polypeptide from the microbial, e.g. , bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g. ,HlyBD. Non- limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g. , hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the polypeptide to be secreted include a "secretion tag" of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g. , OmpT cleavage thereby releasing the

antinflammatory or barrier enhancer molecule(s) into the extracellular milieu. In some embodiments, the secretion system involves the generation of a "leaky" or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl. Lpp functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype.

Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g. , selected from lpp, ompA, ompA, ompF, tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g. , selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[00157] As used herein, the term "conventional treatment" or "conventional therapy" refers to treatment or therapy that is currently accepted, considered current standard of care, and/or used by most healthcare professionals for treating a disease or disorder associated with BCAA. It is different from alternative or complementary therapies, which are not as widely used.

[00158] The articles "a" and "an," as used herein, should be understood to mean

"at least one," unless clearly indicated to the contrary.

[00159] The phrase "and/or," when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and/or C" indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase "and/or" may be used interchangeably with "at least one of or "one or more of the elements in a list.

[00160] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Bacteria

[00161] The genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying deleterious molecules, e.g., chemotherapeutic drugs or metabolites or byproducts thereof, nonsteroidal anti- inflammatory drugs or metabolites or byproducts thereof, or exogenous poisons. In some embodiments, the genetically engineered bacteria are further capable of expressing an anti- inflammatory molecule and/or a gut barrier enhancer molecule.

[00162] In some embodiments, the genetically engineered bacteria are nonpathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some

embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides

thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii,

Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.

[00163] In some embodiments, the genetically engineered bacterium is a Gram- positive bacterium, e.g., Clostridium, that is naturally capable of producing high levels of butyrate. In some embodiments, the genetically engineered bacterium is selected from the group consisting of C. butyricum ZJUCB, C. butyricum S21, C. thermobutyricum ATCC 49875, C. beijerinckii, C. populeti ATCC 35295, C. tyrobutyricum JM1, C. tyrobutyricum CIP 1-776, C. tyrobutyricum ATCC 25755, C. tyrobutyricum CNRZ 596, and C. tyrobutyricum ZJU 8235. In some embodiments, the genetically engineered bacterium is C. butyricum CBM588, a probiotic bacterium that is highly amenable to protein secretion and has demonstrated efficacy in treating IBD (Kanai et al, 2015). In some embodiments, the genetically engineered bacterium is Bacillus, a probiotic bacterium that is highly genetically tractable and has been a popular chassis for industrial protein production; in some embodiments, the bacterium has highly active secretion and/or no toxic byproducts (Cutting, 2011).

[00164] In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell.

[00165] In some embodiments, the genetically engineered bacteria are

Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the

Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors (e.g., E. coli a-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle's therapeutic efficacy and safety have convincingly been proven (Ukena et al., 2007). One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be modified and adapted for other species, strains, and subtypes of bacteria. In some

embodiments, the genetically engineered bacteria are E. coli Nissle and are naturally capable of promoting tight junctions and gut barrier function. In some embodiments, the genetically engineered bacteria are E. coli and are highly amenable to recombinant protein technologies.

[00166] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. It is known, for example, that the clostridial butyrogenic pathway genes are widespread in the genome-sequenced Clostridia and related species (Aboulnaga et al., 2013). Furthermore, genes from one or more different species of bacteria can be introduced into one another, e.g., the butyrogenic genes from Peptoclostridium difficile have been expressed in Escherichia coli (Aboulnaga et al., 2013).

[00167] In one embodiment, the recombinant bacterial cell does not colonize the subject having the disorder. Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum

(Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the genetically engineered bacteria may require continued

administration. Residence time in vivo may be calculated for the genetically engineered bacteria. In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention, e.g. as described herein.

[00168] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009). Thus the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria.

[00169] In certain embodiments, the payload(s) described below are expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the payload is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria.

[00170] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells disclosed herein.

[00171] In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein.

[00172] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) further comprise a kill- switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as

ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

[00173] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s)is an auxotroph. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s)is an auxotroph and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.

[00174] In some embodiments of the above described genetically engineered bacteria, the gene encoding one or more enzyme(s) capable of detoxifying a deleterious moleculeis present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an anti- inflammatory or gut barrier enhancer molecule is present in the bacterial chromosome. In some embodiments, a gene sequence encoding a secretion protein or protein complex, such as any of the secretion systems disclosed herein, for secreting a biomolecule (e.g. an enzyme capable of detoxifying a deleterious molecule, gene sequence encoding an anti- inflammatory molecule, and/or gene sequence encoding a gut barrier enhancer molecule), is present on a plasmid in the bacterium. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome.

Chemotherapeutic drugs

[00175] In some embodiments, the genetically engineered bacteria are capable of inhibiting, metabolizing, and/or detoxifying chemotherapeutic drugs or metabolites or byproducts thereof. In some embodiments, the genetically engineered bacteria detoxify the drug or metabolite or byproduct after the chemotherapeutic drug exerts its therapeutically beneficial effects, e.g., cytotoxicity in cancerous cells. In some embodiments, the genetically engineered bacteria are administered before, together with, and/or after administration of the chemotherapeutic drug. In some embodiments, the genetically engineered bacteria are capable of detoxifying, inhibiting, and/or metabolizing the drug or metabolite or byproduct, thereby reducing chemotherapy-induced diarrhea, reducing chemotherapy-induced toxicity, increasing chemotherapy dosage amount, increasing chemotherapy dosage frequency, and/or increasing chemotherapy efficacy. In some embodiments, the molecule to be detoxified is a

chemotherapeutic drug selected from irinotecan, methotrexate, an antimetabolite, gemcitabine, cytosine arabinoside, a fluoropyrimidine, fluoro uracil, capecitabine, tegafur- uracil, a multitargeted folinic acid antagonist, pemetrexed, raltitrexed, gemcitabine, a plant alkaloid, a vinca alkaloid, vincristine, vinorelbine, a epipodophyllotoxin, etoposide, a taxane, paclitaxel, docetaxel, a topoisomerase I inhibitor, a cytotoxic antibiotic, an anthracycline, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, an alkylating agent, cyclophosphamide, a platinum, cisplatin, carboplatin, oxaliplatin, nedaplatin, an antibody, ipilumumab, an antibody against VEGF, bevacizumab, a tyrosine-kinase inhibitor, an EGFR inhibitor, lapatinib, cetuximab, or a metabolite or byproduct of said chemotherapeutic drug, e.g., SN-38.

Methotrexate

[00176] A chemotherapeutic drug may have therapeutically beneficial effects as well as deleterious effects. For example, methotrexate is an effective antineoplastic agent, but can also cause dose-limiting diarrhea, gastrointestinal toxicity, and bone marrow toxicity (Chabner et al., 1972). Methotrexate is a folate antagonist that acts by inhibiting several enzymes of the folate pathway and disrupting folate homeostasis. In species that possess hepatic enzymes capable of metabolizing methotrexate, e.g., in rabbits, methotrexate is rapidly degraded and virtually no n- toxic (Chabner et al, 1972). In humans and mice, however, no natural enzymes are capable of metabolizing methotrexate; methotrexate persists in the blood for many hours after administration and is capable of causing dose-limiting side effects.

[00177] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate. In some embodiments, the payload is a small molecule that is capable of inhibiting methotrexate.

In some embodiments, the payload is an enzyme that is capable of metabolizing methotrexate into non-toxic metabolites. In certain embodiments, the enzyme capable of metabolizing methotrexate is from a non-human species, e.g., a plant, bacterial, or other mammalian enzyme.

In some embodiments, the enzyme is a synthetic or modified enzyme. In some embodiments, the genetically engineered bacteria comprise a gene encoding carboxypeptidase Gi (CPD Gi) and are capable of detoxifying methotrexate. In certain embodiments, the genetically engineered bacteria comprise a gene encoding Pseudomonas stutzeri CPD Gi (Chabner et ah,

1972). In some embodiments, the genetically engineered bacteria comprise a gene encoding carboxypeptidase G ₂ (CPD G ₂ ) and are capable of detoxifying methotrexate. Exemplary amino acid sequences for CPD Gi and CPD G ₂ are provided below.

[00178] Pseudomonas stutzeri carboxypeptidase CPD Gi

MAS GRDERPPWRLGRLLLLMCLLLLGS S ARAAHIKKAE ATTTTTS AG AE AAEGQFDR

YYHEEELESALREAAAAGLPGLARLFSIGRSVEGRPLWVLRLTAGLGSLIPEGDAGP DA

AGPDAAGPLLPGRPQVKVGNMHGDETVSRQVLIYLARELAAGYRRGDPRLVRLLNTT

D VYLLPS LNPDGFERAREGDCGFGDGGPS GAS GRDNSRGRDLNRS FPDQFSTGEPPAL

DEVPEVRALIEWIRRNKFVLSGNLHGGSVVASYPFDDSPEHKATGIYSKTSDDEVFK YL

AKAYASNHPIMKTGEPHCPGDEDETFKDGITNGAHWYDVEGGMQDYNYVWANCFEI

TLELSCCKYPPASQLRQEWENNRESLITLIEKVHIGVKGFVKDSITGSGLENATISV AGI

NHNITTGRFGDFYRLLVPGTYNLTVVLTGYMPLTVTNVVVKEGPATEVDFSLRPTVT S

VIPDTTEAVSTASTVAIPNILSGTSSSYQPIQPKDFHHHHFPDMEIFLRRFANEYPN ITRL

YS LGKS VES RELY VMEIS DNPG VHEPGEPEFKYIGNMHGNE V VGRELLLNLIE YLC KN

FGTDPEVTDLVHNTRIHLMPSMNPDGYEKSQEGDSISVIGRNNSNNFDLNRNFPDQF V

QITDPTQPETIAVMSWMKSYPFVLSANLHGGSLVVNYPFDDDEQGLATYSKSPDDAV F

QQI ALS YS KENS QMFQGRPC KNM YPNE YFPHGITNG AS WYN VPGGMQD WN YLQTNC

FEVTIELGCVKYPLEKELPNFWEQNRRSLIQFMKQVHQGVRGFVLDATDGRGILNAT IS

VAEINHPVTTYKTGDYWRLLVPGTYKITASARGYNPVTKNVTVKSEGAIQVNFTLVR S

STDSNNESKKGKGASSSTNDASDPTTKEFETLIKDLSAENGLESLMLRSSSNLALAL YR

YHS YKDLS EFLRGLVMN YPHITNLTNLGQS TE YRHIWS LEIS NKPN VS EPEEPKIRFV A

GIHGNAPVGTELLLALAEFLCLNYKKNPAVTQLVDRTRIVIVPSLNPDGRERAQEKD CT

SKIGQTNARGKDLDTDFTNNASQPETKAIIENLIQKQDFSLSVALDGGSMLVTYPYD KP

VQT VENKETLKHL AS LY ANNHPS MHMGQPS CPNKS DENIPGG VMRG AE WHS HLGS M

KD YS VT YGHCPEIT V YTS CC YFPS A ARLPS LW ADNKRS LLS MLVE VHKG VHGFVKD K

TGKPISKAVIVLNEGIKVQTKEGGYFHVLLAPGVHNIIAIADGYQQQHSQVFVHHDA AS

SVVIVFDTDNRIFGLPRELVVTVSGATMSALILTACIIWCICSIKSNRHKDGFHRLR QHH

DE YEDEIRMMS TGS KKS LLS HEFQDETDTEEETLYS S KH (SEQ ID NO: 328) [00179] Pseudomonas sp.carboxypeptidase CPD G ₂

MRPSIHRTAIAAVLATAFVAGTALAQKRDNVLFQAATDEQPAVIKTLEKLVNIETGTG DAEGIAAAGNFLEAELKNLGFTVTRSKSAGLVVGDNIVGKIKGRGGKNLLLMSHMDT VYLKGILAKAPFRVEGDKAYGPGIADDKGGNAVILHTLKLLKEYGVRDYGTITVLFNT DEEKGSFGSRDLIQEEAKLADYVLSFEPTSAGDEKLSLGTSGIAYVQVNITGKASHAGA APELG VN ALVE AS DLVLRTMNIDD KAKNLRFN WTI AKAGN VS NIIP AS ATLN AD VRY A RNEDFDAAMKTLEERAQQKKLPEADVKVIVTRGRPAFNAGEGGKKLVDKAVAYYKE AGGTLGVEERTGGGTDAAYAALSGKPVIESLGLPGFGYHSDKAEYVDISAIPRRLYMA ARLIMDLGAGK (SEQ ID NO: 329)

[00180] In some embodiments, the genetically engineered bacterium comprises one or more genes encoding CPD Gi {i.e., one or more cpgl genes). In some embodiments, the genetically engineered bacteria comprises a CPD Gi gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 328. In some embodiments, the genetically engineered bacteria comprises a CPD Gi gene sequence encoding a polypeptide comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 328. In some

embodiments, the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 328. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 328.

[00181] In some embodiments, the genetically engineered bacterium comprises one or more genes encoding CPD G ₂ {i.e., one or more cpg2 genes). In some embodiments, the genetically engineered bacteria comprises a CPD G ₂ gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 329. In some embodiments, the genetically engineered bacteria comprises a CPD G ₂ gene sequence encoding a polypeptide comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 329. In some

embodiments, the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 329. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 329. [00182] In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of a constitutive promoter. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of an inducible promoter. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the exogenous

environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some embodiments, the exogenous environmental conditions are low- oxygen or anaerobic conditions, such as the environment of the mammalian gut.

[00183] The gene or gene cassette for producing the methotrexate-detoxifying payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the sequence encoding CPD G ₂ may be integrated into the bacterial chromosome. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 24). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.

[00184] In some embodiments, the genetically engineered bacteria produce more of the methotrexate-detoxifying payload, e.g., CPD G ₂ protein or transcript, than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria are capable of reducing local and/or systemic methotrexate levels, e.g., in the gut. In some embodiments, the genetically engineered bacteria are capable of detoxifying methotrexate in the intestinal lumen, and do not affect the therapeutically beneficial

antineoplastic/cytotoxic effects of naproxen on cancerous cells.

[00185] In some embodiments, the genetically engineered bacteria further comprise a gene cassette for producing one or more ant i- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL- 10, IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD ₂ , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S. Application No. 14/998,376, filed December 22, 2015; PCT Application No. PCT/US2016/20530, filed March 2, 2016; U.S. Application No. 15/301,230, filed September 30, 2016; PCT Application No. PCT/US2016/050836, filed September 8, 2016; U.S.

Application No. 15/260,319, filed September 8, 2016; U.S. Provisional Application No.

62/095,415, filed December 22, 2014; and PCT Application No. PCT/US2015/67435, filed December 22, 2015, PCT Application No. PCT/US 2016/039444, and filed on June 24, 2016; PCT Application No. PCT/US2016/069052, filed on December 28, 2016, each of which are incorporated by reference herein intheir entireties, including the drawings. In some specific embodiments, the genetically engineered bacteria encode a biosynthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating methotrexate-induced diarrhea and/or other drug-induced symptom. In some embodiments, the short-chain fatty acid is selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically

engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate and further comprise a gene or gene cassette for producing a payload that is capable of enhancing gut barrier function, e.g., a short-chain fatty acid. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate and further comprise a gene cassette encoding a biosynthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene encoding Pseudomonas stutzeri CPD Gi and further comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically engineered bacteria comprise a gene encoding CPD G ₂ and further comprise a gene cassette encoding a bio synthetic pathway for producing butyrate. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low- copy plasmid, or a chromosome, as described above.

[00186] At higher doses, methotrexate can be used to treat certain types of cancers. At lower doses, methotrexate can be used to treat rheumatic diseases. In certain embodiments, the genetically engineered bacteria of the invention are capable of reducing methotrexate-induced diarrhea and toxicity for non-cancer indications, e.g., rheumatoid arthritis.

Irinotecan

[00187] Alternatively, a chemotherapeutic drug may have therapeutically beneficial effects, but its metabolites or byproducts have deleterious effects. For example, irinotecan is an effective antineoplastic agent, but its metabolite SN-38 can cause dose-limiting diarrhea and gastrointestinal toxicity (Steiner et al., 2010). Irinotecan is a topoisomerase I inhibitor that is used to treat colorectal cancer, non-small cell lung cancer, small cell lung cancer, and pancreatic cancer. Hepatic and peripheral carboxylesterase convert irinotecan to its active metabolite SN-38 (7-ethyl-lO-hydroxycamptothecin). SN-38 is approximately 100- 1,000 times more cytotoxic than irinotecan (Steiner et al, 2010). Hepatic uridine diphosphate glucuronosyltransferase-lAl (UDP-GT 1A1) subsequently glucuronidates SN-38 to the much less toxic metabolite SN-38G. In the intestinal lumen, however, commensal bacteria the produce β-glucuronidase deconjugate SN-38G into toxic SN-38, which can cause intestinal toxicity and diarrhea. Commensal bacteria that are known to have B -glucuronidase activity include enterobacteriaceae, E. coli, Salmonella spp, Shigella spp, Yersinia spp, Citrbacter spp, Hafnia spp, Edwardia spp, Flavobacterium spp, Bacteroids spp, Streptococcus spp,

Corynebacterium spp, and Clostridium spp. Antibiotic treatment dimishes colonic injury following irinotecan administration (Takasuna et al., 1996), and bacterial flora changes in the colon following irinotecan use (Stringer et al., 2007). Irinotecan administration also alters absorptive processes, and fecal sodium and potassium levels rise, leading to diarrhea (Stringer et al., 2007). Thus, irinotecan chemotherapy can cause acute diarrhea, which occurs within 24 hours of receiving the drug, and/or delayed diarrhea, which occurs after 24 hours of receiving the drug (Steiner et al, 2010).

[00188] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and/or SN-38. In some embodiments, the payload is a small molecule that inhibits β-glucuronidase to prevent the conversion of irinotecan into the toxic metabolite SN-38 (Wallace et al., 2010). In some embodiments, the small molecule that inhibits β-glucuronidase is D-saccharic acid 1.4- lactone (SAL) (Fittkau et al., 2004). In some embodiments, the payload is a small molecule that is capable of inhibiting SN-38. In some embodiments, the payload is an enzyme that is capable of metabolizing SN-38 into non-toxic metabolites. In some embodiments, the payload is an enzyme that is capable of glucuroniding SN-38, thereby converting it into non-toxic SN- 38G. In certain embodiments, the enzyme is from a non-human species, e.g., a plant, bacterial, or other mammalian enzyme. In some embodiments, the enzyme is a synthetic or modified enzyme. In some embodiments, the payload is a molecule that is capable of inhibiting or killing the commensal bacteria that produce β-glucuronidase. In some embodiments, the payload is a molecule that promotes changes to the intestinal microflora, e.g., enhancing the survival and/or proliferation of the genetically engineered bacteria, thereby outcompeting commensal bacteria that produce β-glucuronidase. In some embodiments, the genetically engineered bacteria comprise a gene encoding UDP-glucuronsyltransferase, which is capable of adding glucuronic acid to SN-38, and are capable of detoxifying SN-38. To identify relevant payloads, known bacterial metabolites will be surveyed via metagenomic studies and assessed for potency to inhibit bacterial glucouronidase substrate. Bio informatics will be used to inform, select, and stratify physiologically relevant glucouronidase inhibitors. An exemplary amino acid sequence of UDP-glucuronsyltransferase is provided below.

[00189] UDP-glucuronsyltransferase

MARGLQVPLPRLATGLLLLLSVQPWAESGKVLVVPTDGSPWLSMREALRELHARGHQ

AVVLTPEVNMHIKEEKFFTLTAYAVPWTQKEFDRVTLGYTQGFFETEHLLKRYSRSM

AIMNNVSLALHRCCVELLHNEALIRHLNATSFDVVLTDPVNLCGAVLAKYLSIPAVF F

WRYIPCDLDFKGTQCPNPSSYIPKLLTTNSDHMTFLQRVKNMLYPLALSYICHTFSA PY

AS LAS ELFQRE VS V VDLVS Y AS V WLFRGDF VMD YPRPIMPNM VFIGGINC ANGKPLS Q

EFEAYINASGEHGIVVFSLGSMVSEIPEKKAMAIADALGKIPQTVLWRYTGTRPSNL AN

NTILVKWLPQNDLLGHPMTR AFITH AGS HG V YES ICNG VPM VMMPLFGD QMDN AKR

METKGAGVTLNVLEMTSEDLENALKAVINDKSYKENIMRLSSLHKDRPVEPLDLAVF

WVEFVMRHKGAPHLRPAAHDLTWYQYHSLDVIGFLLAVVLTVAFITFKCCAYGYRK

CLGKKGRVKKAHKS KTH (SEQ ID NO: 330)

[00190] In some embodiments, the genetically engineered bacterium comprises one or more genese encoding UDP-glucuronsyltransferase. In some embodiments, the genetically engineered bacteria comprises a UDP-glucuronsyltransferase gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 330. In some embodiments, the genetically engineered bacteria comprises a UDP-glucuronsyltransferase gene sequence encoding a polypeptide comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 330. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 330. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 330.

[00191] In some embodiments, the gene or gene cassette for producing the SN-

38-detoxifying pay load is expressed under the control of a constitutive promoter. In some embodiments, the gene or gene cassette for producing the SN-38-detoxifying payload is expressed under the control of an inducible promoter. In some embodiments, the gene or gene cassette for producing the SN-38-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions. In some embodiments, the gene or gene cassette for producing the SN-38-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g. , propionate. In some embodiments, the exogenous environmental conditions are low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.

[00192] The gene or gene cassette for producing the SN-38-detoxifying payload, e.g. , UDP-glucuronsyltransferase, may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the SN-38-detoxifying payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the SN-38-detoxifying payload is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the SN-38-detoxifying payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 24. The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.

[00193] In some embodiments, the genetically engineered bacteria produce more of the SN-38-detoxifying pay load than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria are capable of reducing local and/or systemic SN-38 levels, e.g., in the gut. In some embodiments, the genetically engineered bacteria are capable of detoxifying SN-38 in the intestinal lumen, and do not affect the therapeutically beneficial antineoplastic/cytotoxic effects of irinotecan on cancerous cells.

[00194] In some embodiments, the genetically engineered bacteria further comprise a gene cassette for producing one or more ant i- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL- 10, IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD ₂ , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S. Application No. 14/998,376, filed December 22, 2015; PCT Application No. PCT/US2016/20530, filed March 2, 2016; U.S. Application No. 15/301,230, filed September 30, 2016; PCT Application No. PCT/US2016/050836, filed September 8, 2016; U.S.

Application No. 15/260,319, filed September 8, 2016; U.S. Provisional Application No.

62/095,415, filed December 22, 2014; and PCT Application No. PCT/US2015/67435, filed December 22, 2015, PCT Application No. PCT/US 2016/039444, and filed on June 24, 2016; PCT Application No. PCT/US2016/069052, filed on December 28, 2016, the entire contents of each of which are expressly incorporated herein by reference. In some specific embodiments, the genetically engineered bacteria encode a bio synthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating irinotecan- induced diarrhea and/or other drug-induced symptom. In some

embodiments, the short-chain fatty acid is selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically

engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and further comprise a gene or gene cassette for producing a payload that is capable of enhancing gut barrier function, e.g. , a short-chain fatty acid. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and further comprise a gene cassette encoding a bio synthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene encoding UDP-glucuronsyltransferase and further comprise a gene cassette encoding a bio synthetic pathway for producing butyrate. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low- copy plasmid, or a chromosome, as described above.

NSAIDs

[00195] In some embodiments, the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying NSAIDs or metabolites or byproducts thereof. In some embodiments, the genetically engineered bacteria detoxify the NSAID or metabolite or byproduct after the NSAID exerts its therapeutically beneficial effects, e.g. , reducing inflammation. In some embodiments, the genetically engineered bacteria are administered before, together with, and/or after NSAID administration (see, e.g., Cohen et al., 2013). In some embodiments, the genetically engineered bacteria are capable of detoxifying, inhibiting, and/or metabolizing the toxic NSAID or metabolite or byproduct, thereby reducing NSAID-induced diarrhea, reducing NSAID-induced toxicity, increasing NSAID dosage amount, increasing NSAID dosage frequency, and/or increasing NSAID efficacy. In some embodiments, the molecule to be detoxified is a NSAID selected from naproxen, indomethacin, ketoprofen, piroxicam, ibuprofen, diclofenac, a COX-2 inhibitor, or a metabolite or byproduct thereof. An exemplary NSAID is described below.

[00196] Many NSAIDs, including naproxen, inhibit the activity of

cyclooxygenase- 1 (COX- 1) and cyclooxygenase-2 (COX-2). The ant i- inflammatory and analgesic effects of NSAIDs are thought to be due to inhibition of COX-2, while the gastrointestinal toxicity of NSAIDs is thought to be due to the inhibition of COX- 1 (Scarpignato 2008). NSAID-induced toxicity to the intestinal epithelium occurs after oral administration. Local sub-cellular damage occurs at the brush border cell member upon entry of the typically acidic NSAID. Mitochondrial oxidative phosphorylation is disrupted, leading to ATP deficiency, increased mucosal permeability, and entrance of luminal molecules such as bacteria, pancreatic juice, bile acids, and dietary macromolecules, which causes an

inflammatory response.

[00197] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying one or more NSAIDs, e.g. , naproxen. In some embodiments, the payload is a small molecule that is capable of inhibiting one or more NSAIDs, e.g. , naproxen. In some embodiments, the payload is a proton pump inhibitor, and the genetically engineered bacteria are capable of ameliorating NSAID-induced intestinal damage (see, e.g., Scarpignato, 2008). In some embodiments, the payload is an enzyme that is capable of metabolizing the NSAID into non-toxic metabolites. In certain embodiments, the enzyme capable of metabolizing the NSAID is from a non-human species, e.g. , a plant, bacterial, or other mammalian enzyme. In some embodiments, the enzyme is a synthetic or modified enzyme. B -glucuronidase inhibition can alleviate NSAID- induced enteropathy (LoGuidice et al., 2012). In some embodiments, the genetically engineered bacteria comprise a gene encoding glucuronosyltransferase (SEQ ID NO: 331) and are capable of glucuronidating and detoxifying naproxen. In some embodiments, the genetically engineered bacteria comprise a gene encoding glucuronosyltransferase (SEQ ID NO: 331), a gene cassette for producing butyrate, and are capable of detoxifying naproxen. An exemplary amino acid sequence of glucuronosyltransferase is provided below.

[00198] Glucuronosyltransferase

MACLLRSFQRISAGVFFLALWGMVVGDKLLVVPQDGSHWLSMKDIVEVLSDRGHEIV

VVVPEVNLLLKESKYYTRKIYPVPYDQEELKNRYQSFGNNHFAERSFLTAPQTEYRN N

MIVIGLYFINCQSLLQDRDTLNFFKESKFDALFTDPALPCGVILAEYLGLPSVYLFR GFP

CS LEHTFS RS PDP VS YIPRC YTKFS DHMTFS QRV ANFLVNLLEP YLFYCLFS KYEEL AS A

VLKRDVDIITLYQKVSVWLLRYDFVLEYPRPVMPNMVFIGGINCKKRKDLSQEFEAY I

NASGEHGIVVFSLGSMVSEIPEKKAMAIADALGKIPQTVLWRYTGTRPSNLANNTIL VK

WLPQNDLLGHPMTRAFITHAGSHGVYESICNGVPMVMMPLFGDQMDNAKRMETKG

AGVTLNVLEMTSEDLENALKAVINDKSYKENIMRLSSLHKDRPVEPLDLAVFWVEFV

MRHKGAPHLRPAAHDLTWYQYHSLDVIGFLLAVVLTVAFITFKCCAYGYRKCLGKK

GRVKKAHKS KTH (SEQ ID NO: 331)

[00199] In some embodiments, the genetically engineered bacterium comprises one or more genes encoding glucuronosyltransferase. In some embodiments, the genetically engineered bacteria comprises a glucuronosyltransferase gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 331. In some embodiments, the genetically engineered bacteria comprises a glucuronosyltransferase gene sequence encoding a polypeptide

comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 331. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 331. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 331.

[00200] Some studies have shown that decreased Gram-negative but not Gram- positive organisms lessens indomethacin-induced intestinal damage (Scarpignato, 2008). In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that promotes changes to the intestinal microflora, e.g. , enhancing the survival and/or proliferation of Gram-positive bacteria. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of inhibiting or killing Gram-negative bacteria.

[00201] In some embodiments, the gene or gene cassette for producing the

NSAID-detoxifying payload is expressed under the control of a constitutive promoter. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is expressed under the control of an inducible promoter. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g. , propionate. In some embodiments, the exogenous environmental conditions are low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.

[00202] The gene or gene cassette for producing the NSAID-detoxifying payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 24). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.

[00203] In some embodiments, the genetically engineered bacteria produce more of the NSAID-detoxifying payload than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria are capable of reducing local and/or systemic NSAID levels, e.g., in the gut. In some embodiments, the genetically engineered bacteria are capable of detoxifying the NSAID in the intestinal lumen, and do not affect the therapeutically beneficial anti- inflammatory effects of the NSAID systemically.

[00204] In some embodiments, the genetically engineered bacteria further comprise a gene cassette for producing one or more anti- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL- 10, IL-27, TGF-βΙ, TGF-β2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD ₂ , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S. Application No. 14/998,376, filed December 22, 2015; PCT Application No. PCT/US2016/20530, filed March 2, 2016; U.S. Application No. 15/301,230, filed September 30, 2016; PCT Application No. PCT/US2016/050836, filed September 8, 2016; U.S.

Application No. 15/260,319, filed September 8, 2016; U.S. Provisional Application No.

62/095,415, filed December 22, 2014; and PCT Application No. PCT/US2015/67435, filed December 22, 2015, PCT Application No. PCT/US 2016/039444, and filed on June 24, 2016; PCT Application No. PCT/US2016/069052, filed on December 28, 2016. In some specific embodiments, the genetically engineered bacteria encode a biosynthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating NSAID-induced diarrhea and/or other drug-induced symptom. In some embodiments, the short-chain fatty acid is selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying the NSAID and further comprise a gene or gene cassette for producing a payload that is capable of enhancing gut barrier function, e.g., a short-chain fatty acid. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying the NSAID and further comprise a gene cassette encoding a biosynthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene encoding glucuronosyltransferase and further comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low- copy plasmid, or a chromosome, as described above.

Heavy metals

[00205] In some embodiments, the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying one or more heavy metals, thereby ameliorating one or more symptoms of heavy metal poisoning. In some embodiments, the genetically engineered bacteria of the invention are capable of ameliorating acute heavy metal poisoning and/or chronic heavy metal poisoning. In some embodiments, genetically engineered bacteria of the invention are capable of ameliorating one or more symptoms of aluminum poisoning, antimony poisoning, arsenic poisoning, barium poisoning, bismuth poisoning, cadmium poisoning, chromium poisoning, cobalt poisoning, copper poisoning, gold poisoning, iron poisoning, lead poisoning, lithium poisoning, manganese poisoning, mercury poisoning, nickel poisoning, phosphorous poisoning, platinum poisoning, selenium poisoning, silver poisoning, thallium poisoning, tin poisoning, and/or zinc poisoning. Examples of heavy metal toxicity linked to bacteria are shown in Table 2. Table 2. Heavy metal link with bacteria/probiotics

[00206] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering a heavy metal. In some embodiments, the payload is a heavy metal chelator. In some embodiments, the payload is a plant phytochelatin. In certain embodiments, the payload is from a non-human species, e.g. , a plant, bacterial, or other mammalian molecule. In some embodiments, the payload is a synthetic or modified molecule.

[00207] In some embodiments, the genetically engineered bacteria are capable of expressing plant phytochelatins, particularly on the surface of the bacteria (see, e.g., Bae et al., 2000). Thus, the genetically engineered bacteria of the invention are capable of binding to cadmium, thereby ameliorating one or more symptoms of cadmium poisoning. In some embodiments, the genetically engineered bacteria comprise a gene encoding a plant phytochelatin and a gene cassette for producing butyrate. In some embodiments, the genetically engineered bacteria comprise a gene encoding an enzyme involved in the production of a plant phytochelatin. Exemplary plant phytochelatins include, but are not limited to, glutathione, homoglutathione, hydroxymethylglutathione, and

glutamylcysteinylglutamate. [00208] In some embodiments, the gene or gene cassette for producing the metal- binding payload, e.g., a plant phytochelatin, is expressed under the control of a constitutive promoter. In some embodiments, the gene or gene cassette for producing the metal-binding payload, e.g., a plant phytochelatin, is expressed under the control of an inducible promoter. In some embodiments, the gene or gene cassette for producing the metal-binding payload is expressed under the control of a promoter that is induced by exogenous environmental conditions. In some embodiments, the gene or gene cassette for producing the metal-binding payload is expressed under the control of a promoter that is induced by exogenous

environmental conditions specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some embodiments, the exogenous environmental conditions are low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.

[00209] The gene or gene cassette for producing the metal-binding payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the metal-binding payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the metal-binding payload is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the metal- binding payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used. The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.

[00210] In some embodiments, the genetically engineered bacteria produce more of the metal-binding payload than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria are capable of reducing local and/or systemic heavy metal levels. [00211] In some embodiments, the genetically engineered bacteria further comprise a gene cassette for producing one or more ant i- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP- 1, IL- 10, IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD ₂ , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S. Application No. 14/998,376, filed December 22, 2015; PCT Application No. PCT/US2016/20530, filed March 2, 2016; U.S. Application No. 15/301,230, filed September 30, 2016; PCT Application No. PCT/US2016/050836, filed September 8, 2016; U.S.

Application No. 15/260,319, filed September 8, 2016; U.S. Provisional Application No.

62/095,415, filed December 22, 2014; and PCT Application No. PCT/US2015/67435, filed December 22, 2015, PCT Application No. PCT/US 2016/039444, and filed on June 24, 2016; PCT Application No. PCT/US2016/069052, filed on December 28, 2016, the entire contents of which are expressly incorporated herein by reference. In some specific embodiments, the genetically engineered bacteria encode a biosynthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating heavy metal- induced diarrhea and/or other drug-induced symptom. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering one or more heavy metals and further comprise a gene or gene cassette for producing a payload that is capable of reducing inflammation and/or enhancing gut barrier function, e.g. , a short-chain fatty acid. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering one or more heavy metals and further comprise a gene cassette encoding a biosynthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate. In some embodiments, the genetically engineered bacteria comprise a gene encoding a plant phytochelatin and further comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, an inducible promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above. The gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome, as described above.

Other Molecules

[00212] Recent investigations have revealed that pharmaceuticals including antibiotics, anti-convulsants, mood stabilizers and sex hormones have been found in the drinking water supplies of at least 41 million Americans. Accordingly, in some embodiments, the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying one or more environmental toxic molecules, such as antibiotics, anticonvulsants, mood stabilizers and sex hormones, e.g., estrogen.

[00213] It has been recognized that Pseudomonas produces many enzymes that can degrade a wide array of organic compounds including drugs. Therefore, Pseudomonas can be used to identify enzymes that can degrade toxic molecules of interest and geneically engineered bacteria comprising genes encoding these enzymes can be made and used as therapeutics.

Anti-inflammation and/or gut barrier function enhancer molecules

[00214] The genetically engineered bacteria comprise one or more gene sequence(s) and/or gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene sequence(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene sequences are multiple copies of the same gene. In some emodiments, the two or more gene sequences are sequences encoding different genes. In some emodiments, the two or more gene sequences are sequences encoding multiple copies of one or more different genes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene cassettes are multiple copies of the same gene cassette. In some emodiments, the two or more gene cassettes are different gene cassettes for producing either the same or different anti- inflammation and/or gut barrier function enhancer molecule(s). In some emodiments, the two or more gene cassettes are gene cassettes for producing multiple copies of one or more different anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the anti- inflammation and/or gut barrier function enhancer molecule is selected from the group consisting of a short-chain fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP- 1, IL- 10 (human or viral), IL-27, TGF-βΙ, TGF-P2, N-acylphosphatidylethanolamines (NAPEs), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, kynurenine, typtophan metabolite, indole, indole metabolite, a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-γ, IL- Ιβ, IL-6, IL-8, IL- 17, and/or chemokines, e.g., CXCL-8 and CCL2, AHR agonist (e.g., indole acetic acid, indole-3-aldehyde, and indole), PXR agonist (e.g., IP A), HDAC inhibitor (e.g., butyrate), GPR41 and/or GPR43 activator (e.g., butyrate and/or propionate and/or acetate), GPR109A activator (e.g. , butyrate), inhibitor of NF-kappaB signaling (e.g. , butyrate), modulator of PPARgamma (e.g. , butyrate), activator of AMPK signaling (e.g. , acetate), modulator of GLP- 1 secretion, and hydroxyl radical scavengers and antioxidants (e.g., IP A). A molecule may be primarily anti- inflammatory, e.g. , IL- 10, or primarily gut barrier function enhancing, e.g. , GLP- 2. Alternatively, a molecule may be both anti- inflammatory and gut barrier function

enhancing.

[00215] In some embodiments, the genetically engineered bacteria of the invention express one or more anti- inflammation and/or gut barrier function enhancer molecule(s) that is encoded by a single gene, e.g. , the molecule is elafin and encoded by the PI3 gene, or the molecule is inter leukin- 10 and encoded by the IL10 gene. In alternate embodiments, the genetically engineered bacteria of the invention encode one or more an anti- inflammation and/or gut barrier function enhancer molecule(s), e.g. , butyrate, that is

synthesized by a bio synthetic pathway requiring multiple genes.

[00216] The one or more gene sequence(s) and/or gene cassette(s) may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some

embodiments, expression from the plasmid may be useful for increasing expression of the anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the gene sequence(s)or gene cassette(s) for producing the anti- inflammation and/or gut barrier function enhancer molecule(s) is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the butyrate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle:

malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g. , Fig. 51 for exemplary insertion sites). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.

Short chain Fatty Acids and Tryptophan Metabolites

[00217] One strategy in the treatment, prevention, and/or management of disorders may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti- inflammatory effectors.

[00218] For example, leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate, and those derived from tryptophan metabolism. These metabolites have been shown to play a major role in the prevention of inflammatory disease. As such one approach in the treatment, prevention, and/or management of gut barrier health may be to provide a treatment which contains one or more of such metabolites.

[00219] For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity (e.g. , as reviewed in

Thorburn et al., Diet, Metabolites, and "Western- Lifestyle" Inflammatory Diseases; Immunity Volume 40, Issue 6, 19 June 2014, Pages 833-842). (A) SCFA-induced promotion of mucus by gut epithelial cells, possibly through signaling through metabolite sensing GPCRs; (B) SCFA- induced secretion of IgA by B cells; (C) SCFA-induced promotion of tissue repair and wound healing; (D) SCFA-induced promotion of Treg cell development in the gut in a process that presumably facilitates immunological tolerance; (E) SCFA- mediated enhancement of epithelial integrity in a process dependent on inflammasome activation (e.g. , via NALP3) and IL- 18 production; and (F) anti- inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, 11-6, and IFN-gamma), and inhibition of NF-κΒ. Many of these actions of SCFAs in gut homeostatis can be ascribed to GPR43 and GPR109A, which are expressed by the colonic epithelium, by inflammatory leukocytes (e.g. neutrophils and marcophages) and by Treg cells. These receptors signal through G proteins, coupled to MAPK, PI3K and mTOR, as well as a separate arrestin- pathway, leading to NFkappa B inhibition. Other effects can be ascribed to SCFA-mediated HDAC inhibition, e.g. butyrate, which may regulate macrophage function and promote TReg cells.

[00220] In addition, a number of trptophan metabolites, including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehhyde, indole-3 propionic acid, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown to be essential for gut homeostais and promote gut-barrier health. These metabolites bind to aryl hydrocarbon receptor (Ahr). After agonist binding, AhR translocates to the nucleus, where it forms a heterodimer with AhR nuclear translocator (ARNT). AhR-dependent gene expression includes genes involved in the production of mediators important for gut homeostasis; these mediators include IL-22, antimicrobicidal factors, increased Thl7 cell activity, and the maintenance of intraepithelial lymphocytes and RORyt-i- innate lymphoid cells.

[00221] Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3- dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis.

[00222] In addition, some indole metabolites, e.g. , indole 3-propionic acid (IP A), may exert their effect an acitvating ligand of Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function, through downregulation of TLR4 signaling (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate

Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). As a result, indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health.

[00223] Thus, in some embodiments, the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryprophan metabolites Butyrate

[00224] In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes described herein. Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to,

Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some

embodiments, the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from Peptoclostridium difficile, e.g., Peptoclostridium difficile strain 630: bcd.2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. In some embodiments, the genetically engineered bacteria comprise a

combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate. For example, in some embodiments, the genetically engineered bacteria comprise bcd.2, etfB3, etfA3, and thiAl from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.

Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB {e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.n some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

[00225] In some embodiments, additional genes may be mutated or knocked out, to further increase the levels of butyrate production. Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

Compositions Comprising Combinations of Detox Effectors and Gut Barrier Enhancers

[00226] In some embodiments, the bacterial cell produces a first payload that is capable of detoxifying a deleterious molecule. In some embodiments, the bacterial cell produces a second payload that is capable of enhancing gut barrier function and anti- inflammation.

[00227] In some embodiments, the first payload is carboxypeptidase Gi (CPD

Gi) or carboxypeptidase G ₂ (CPD G ₂ ). In some embodiments, the payload is D-saccharic acid 1, 4-lactone (SAL). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen.. In some embodiments, the payload is a proton pump inhibitor. Proton pump inhibitors include, but are not limited to, omeprazole, aspirin, lansoprazole, dexlansoprazole, rabeprazole, pantoprazole, esomeprazole, esomeproazole magnesium/naproxen, and omeprazole/sodium bicarbonate. In some embodiments, the payload is a heavy metal chelator. Heavy metal chelators include, but are not limited to, dimercaprol, dimercapto succinic acid (DMSA), dimercapto-propane sulfonate (DMPS), penicillamine, ethylenediamine tetraacetic acid (calcium disodium versenate) (CaNa2-EDTA), deferoxamine and deferasirox. In some embodiments, the payload is a plant phytochelatin. In some embodiments, the payload is a short-chained fatty acid, e.g. butyrate, propionate, or acetate. In some embodiments, the payload is the enzyme

Pseudomonas.

[00228] In some embodiments, the second payload is butyrate, propionate, acetate, IL-10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, IFN-γ, TNF-a, 1L-1B, or tryptophan and/or its metabolites.

[00229] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00230] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g.,IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00231] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D- saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4- lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00232] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10- hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g.,, IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00233] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , TNF-α. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00234] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in

combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in

combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00235] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor) in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, t a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00236] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00237] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant

phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00238] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00239] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00240] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00241] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., 1L-1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00242] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., 1L-1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00243] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., Ih-Π. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00244] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00245] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D-saccharic acid 1, 4-lactone (SAL), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co -administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4- lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00246] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan. [00247] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00248] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g.,, IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00249] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan. [00250] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00251] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan. [00252] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00253] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00254] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00255] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is coacetatered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. propionate, and is coacetatered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00256] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-10. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-19. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L-1B. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

Inducible Promoters

[00257] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.

[00258] In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.

[00259] In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the tumor microenvironment. In some

embodiments, the promoter is directly or indirectly induced by a molecule that is coadministered with the bacterial cell. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the tumor

microenvironment. In some embodiments, the promoter may be tumor-specific, e.g. , hypoxia inducible. In some embodiments, the promoter may be tissue- specific.

[00260] In certain embodiments, the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In certain embodiments, the bacterial cell comprises one or more gene sequence(s) for producing the payload(s), e.g., gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more antiinflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s), which is operably linked to an oxygen level-dependent promoter such that the therapeutic molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, in low-oxygen conditions, the oxygen level-dependent promoter is activated by a corresponding oxygen level- sensing transcriptional regulator, thereby driving production of the therapeutic molecule(s.). In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) for producing one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s)expressed under the control of a fumarate and nitrate reductase regulator (FNR)-responsive promoter, an anaerobic regulation of arginine deiminiase and nitrate reduction (ANR)-responsive promoter, or a dissimilatory nitrate respiration regulator (DNR)-responsive promoter, which are capable of being regulated by the transcription factors FNR, ANR, or DNR, respectively. In E. coli, FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive.

[00261] FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.

Table 3. FNR Promoter Sequences

[00262] FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.

[00263] As used herein the term "payload" refers to one or more molecules produced by a genetically engineered bacterium, e.g., one or more molecules capable of detoxifying a deleterious molecule, producing one or more anti- inflammatory molecule(s), and/or producing one or more gut barrier enhancer molecule(s), including but not limited to, butyrate, propionate, acetate, IL10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, and/or tryptophan and/or its metabolites. As used herein, the term "polypeptide of interest" or "polypeptides of interest", "protein of interest", "proteins of interest", "payload", "payloads" includes any or a plurality of any of the enzymes capable of detoxifying a deleterious molecule, short chain fatty acid producing enzymes, tryptophan metabolite producing enzymes, enzymes producing any gut barrier enhancer and/or anti- inflammatory metabolite, metabolite

transporters or exporters, detox enzymes and/or any other enzyme(s) described herein. As used herein, the term "gene of interest" or "gene sequence of interest" includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more enzymes capable of detoxifying a deleterious molecule, short chain fatty acid producing enzymes, tryptophan metabolite producing enzymes, enzymes producing any gut barrier enhancer and/or anti- inflammatory metabolite, metabolite transporters or exporters, detox enzymes and/or any other enzyme(s) described herein.

[00264] In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable gene or gene cassette for producing an anti- inflammation and/or gut barrier function enhancer molecule. Non- limiting FNR promoter sequences are provided in Table 4. Table 4 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 6, SEQ ID NO: 7, nirB l promoter (SEQ ID NO: 8), nirB2 promoter (SEQ ID NO: 9), nirB3 promoter (SEQ ID NO: 10), ydfZ promoter (SEQ ID NO: 11), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 12), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 13), fnrS, an anaerobically induced small RNA gene (fnrS l promoter SEQ ID NO: 14 or fnrS2 promoter SEQ ID NO: 15), nirB promoter fused to a crp binding site (SEQ ID NO: 16), and fnrS fused to a crp binding site (SEQ ID NO: 17).

[00265] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, or a functional fragment thereof.

[00266] In one embodiment, the FNR responsive promoter comprises SEQ ID

NO: l. In another embodiment, the FNR responsive promoter comprises SEQ ID NO:2. In another embodiment, the FNR responsive promoter comprises SEQ ID NO:3. In another embodiment, the FNR responsive promoter comprises SEQ ID NO:4. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO:5.

[00267] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al, 2010) or ANR (Ray et ah, 1997). In these embodiments, expression of the payload gene is particularly activated in a low- oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut. In one embodiment, the tumor is a human tumor.

[00268] In other embodiments, the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Gorke and Stiilke, 2008). In some embodiments, the gene or gene cassette for producing an anti- inflammation and/or gut barrier function enhancer molecule is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter {e.g., an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing an payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g., by adding glucose to growth media in vitro.

[00269] In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level- sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level- sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen- level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions.

[00270] In certain embodiments, the non-native oxygen- level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et ah, 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.

[00271] In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated

transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity {see, e.g., Moore et al, (2006). In some embodiments, both the oxygen level- sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions.

[00272] In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the gene encoding the payload are present on different plasmids.

[00273] In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing

transcriptional regulator and the gene encoding the payload are present on the same

chromosome.

[00274] In some instances, it may be advantageous to express the oxygen level- sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some

embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region.

[00275] In some embodiments, the gene or gene cassette for producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by low-oxygen conditions. In some embodiments, the gene or gene cassette for producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present in the chromosome and operably linked to a promoter that is induced by low-oxygen conditions. In some embodiments, the gene or gene cassette for producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some

embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[00276] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene(s) or gene cassette(s) capable of producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s), such that the gene(s) or gene cassette(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, a bacterium may comprise multiple copies of the gene or gene cassette for producing the payload molecule(s). In some embodiments, the gene or gene cassette is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low- copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene or gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing gene or gene cassette expression. In some embodiments, gene or gene cassette is expressed on a chromosome.

[00277] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) or gene cassette(s) capable of producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s). In some embodiments, the gene(s) or gene cassette(s) capable of producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to an oxygen level-dependent promoter. In some embodiments, the gene(s) or gene cassette(s) capable of producing the payload, e.g. , gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) present in a chromosome and operably linked to an oxygen level-dependent promoter.

[00278] In some embodiments, the genetically engineered bacteria produce at least one enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) in low-oxygen conditions to detoxify the toxic molecule and/or to reduce local gut inflammation by at least about 1.5-fold, at least about 2- fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800- fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions.

Inflammation may be measured by methods known in the art, e.g. , counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Meso scale, Cayman Chemical, Qiagen).

[00279] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of one more more payload(s), e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) in low-oxygen conditions than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the enzyme capable of detoxifying a deleterious molecule, anti- inflammation and/or gut barrier enhancer molecule. In embodiments using genetically modified forms of these bacteria, the enzyme capable of detoxifying a deleterious molecule, anti- inflammation and/or gut barrier enhancer molecule will be detectable in low-oxygen conditions.

[00280] In certain embodiments, the anti- inflammation and/or gut barrier enhancer molecule is butyrate. Methods of measuring butyrate levels, e.g. , by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate

embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μΜ/OD, at least about 10 μΜ/OD, at least about 100 μΜ/OD, at least about 500 μΜ/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in low-oxygen conditions.

[00281] In certain embodiments, the anti- inflammation and/or gut barrier enhancer molecule is propionate. Methods of measuring propionate levels, e.g. , by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Hillman, 1978; Lukovac et al., 2014). In some embodiments, measuring the activity and/or expression of one or more gene products in the propionate gene cassette serves as a proxy measurement for propionate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure propionate production. In alternate embodiments, propionate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 μΜ, at least about 10 μΜ, at least about 100 μΜ, at least about 500 μΜ, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, or at least about 50 mM of propionate in low-oxygen conditions.

Inflammatory-Dependent Regulation

RNS -dependent regulation

[00282] In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload, e.g., a detoxification molecule, an antiinflammatory molecule, and/or a gut barrier enhancer molecule, that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory- dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.

[00283] As used herein, "reactive nitrogen species" and "RNS" are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO*), peroxynitrite or peroxynitrite anion (ONOO- ), nitrogen dioxide (·Ν02), dinitrogen trioxide (N203), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOC02-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

[00284] As used herein, "RNS-inducible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS- inducible regulatory region in the absence of RNS ; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g. , a payload gene sequence(s), e.g., any of the payloads described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS- inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

[00285] As used herein, "RNS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more RNS -sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

[00286] As used herein, "RNS-repressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS- repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

[00287] As used herein, a "RNS -responsive regulatory region" refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 5. Table 5. Examples of RNS-sensing transcription factors and RNS-responsive genes

[00288] In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated.

[00289] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a

conformational change, thereby inducing downstream gene expression.

[00290] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR "is an NO- responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide" (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al, 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload.

[00291] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) "promotes the expression of the nir, the nor and the nos genes" in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

[00292] In some embodiments, the tunable regulatory region is a RNS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[00293] In some embodiments, the tunable regulatory region is a RNS- derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is "an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism" (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes. In the presence of RNS, an NsrR

transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s).

[00294] In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g. , from is Neisseria gonorrhoeae, wherein the Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[00295] In some embodiments, the tunable regulatory region is a RNS- repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[00296] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g. , encoding a payload. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g. , a payload gene or genes is expressed. [00297] A RNS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and

corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS- sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS -responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al, 2009; Dunn et al, 2010; Vine et al, 2011;

Karlinsey et al, 2012).

[00298] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[00299] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[00300] In some embodiments, the genetically engineered bacteria comprise a

RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g. , NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g. , NsrR, is deleted or mutated to reduce or eliminate wild- type activity.

[00301] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS- sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

[00302] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g. , the NsrR gene, and a corresponding regulatory region, e.g. , a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS -responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g. , NsrR, that is mutated relative to the wild- type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS.

[00303] In some embodiments, the gene or gene cassette for producing a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[00304] In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more encoding a payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

[00305] In some embodiments, the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to detoxify a toxic molecule and/or reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold as compared to unmodified bacteria of the same subtype under the same conditions.

Inflammation may be measured by methods known in the art, e.g. , counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Meso scale, Cayman Chemical, Qiagen). [00306] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1, 000-fold, or at least about 1, 500-fold more of pay load in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.

[00307] In certain embodiments, the anti- inflammation and/or gut barrier enhancer molecule is butyrate. Methods of measuring butyrate levels, e.g. , by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g. , Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate

embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μΜ/OD, at least about 10 μΜ/OD, at least about 100 μΜ/OD, at least about 500 μΜ/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in the presence of RNS.

ROS-dependent regulation

[00308] In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a payload, e.g., detoxification molecule, an anti- inflammatory molecule, and/or a gut barrier enhancer molecule, that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the payload is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.

[00309] As used herein, "reactive oxygen species" and "ROS" are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal- catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H202), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical (·ΟΗ), superoxide or superoxide anion (·02-), singlet oxygen (102), ozone (03), carbonate radical, peroxide or peroxyl radical (·02-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOCl), nitric oxide (NO*), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al, 2014).

[00310] As used herein, "ROS-inducible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS- inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g., a sequence or sequences encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

[00311] As used herein, "ROS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

[00312] As used herein, "ROS-repressible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

[00313] As used herein, a "ROS -responsive regulatory region" refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 6.

Table 6. Examples of ROS-sensing transcription factors and ROS-responsive genes

[00314] In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated.

[00315] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression. [00316] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR "functions primarily as a global regulator of the peroxide stress response" and is capable of regulating dozens of genes, e.g., "genes involved in H202 detoxification (katE, ahpCF), heme

biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)" and "OxyS, a small regulatory RNA" (Dubbs et al., 2012). The genetically engineered bacteria may comprise any suitable ROS -responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a payload gene. In the presence of ROS, e.g., H202, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked payload gene and producing the payload. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROS- inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.

[00317] In alternate embodiments, the tunable regulatory region is a ROS- inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is "activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression" (Koo et al., 2003). "SoxR is known to respond primarily to superoxide and nitric oxide" (Koo et al., 2003), and is also capable of responding to H202. The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al, 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS- inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing the a payload.

[00318] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette. [00319] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR "binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event," but oxidized OhrR is "unable to bind its DNA target" (Duarte et al., 2010). OhrR is a "transcriptional repressor [that] ... senses both organic peroxides and NaOCl" (Dubbs et al., 2012) and is "weakly activated by H202 but it shows much higher reactivity for organic hydroperoxides" (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g. , a payload gene. In the presence of ROS, e.g. , NaOCl, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing the a payload.

[00320] OhrR is a member of the MarR family of ROS -responsive regulators.

"Most members of the MarR family are transcriptional repressors and often bind to the - 10 or - 35 region in the promoter causing a steric inhibition of RNA polymerase binding" (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g. , Dubbs et al., 2012).

[00321] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is "a MarR-type transcriptional regulator" that binds to an "18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA" and is "reversibly inhibited by the oxidant H202" (Bussmann et al., 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to "a putative polyisoprenoid-binding protein (cgl322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cgl426), two putative FMN reductases (cgl l50 and cgl850), and four putative monooxygenases (cg0823, cgl848, cg2329, and cg3084)" (Bussmann et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS- responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS- derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., a payload. In the presence of ROS, e.g., H202, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload.

[00322] In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[00323] In some embodiments, the tunable regulatory region is a ROS- repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[00324] In some embodiments, the tunable regulatory region is a ROS- repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR "when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)" (Marinho et al., 2014). PerR is a "global regulator that responds primarily to H202" (Dubbs et al., 2012) and "interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes" (Marinho et al, 2014). PerR is capable of binding a regulatory region that "overlaps part of the promoter or is immediately downstream from it" (Dubbs et al., 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et ah, 2012).

[00325] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload, e.g., detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR- repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed.

[00326] A ROS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although "OxyR is primarily thought of as a transcriptional activator under oxidizing conditions... OxyR can function as either a repressor or activator under both oxidizing and reducing conditions" (Dubbs et al., 2012), and OxyR "has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)" (Zheng et al., 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS- responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et al., 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by RosR. In addition, "PerR- mediated positive regulation has also been observed... and appears to involve PerR binding to distant upstream sites" (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS- responsive regulatory region from a gene that is activated by PerR.

[00327] One or more types of ROS-sensing transcription factors and

corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, "OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both" (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS -responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.

[00328] Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 7. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NOs: 18, 19, 20, or 21, or a functional fragment thereof. Table 7. Nucleotide sequences of exemplary OxyR-regulated regulatory regions

[00329] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the payload molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the payload molecule. In some embodiments, the ROS- sensing transcription factor and payload molecule, e.g., therapeutic molecule, are divergently transcribed from a promoter region.

[00330] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[00331] In some embodiments, the genetically engineered bacteria comprise a

ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROS- sensing transcription factor, e.g. , OxyR, is deleted or mutated to reduce or eliminate wild-type activity.

[00332] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS- sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

[00333] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g. , the soxR gene, and a corresponding regulatory region, e.g. , a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS -responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g. , OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of ROS, as compared to the wild- type transcription factor under the same conditions. In some embodiments, both the ROS- sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS.

[00334] In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some

embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some

embodiments, expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[00335] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s). In some embodiments, the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS -responsive regulatory region. In some embodiments, the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS -responsive regulatory region.

[00336] Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor.

[00337] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g. , in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the payload. In some embodiments, the gene encoding the payload is expressed on a low-copy plasmid. In some embodiments, the low- copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non- inducing conditions. In some embodiments, the gene encoding the payload is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the payload. In some embodiments, the gene encoding the payload is expressed on a chromosome.

[00338] In some embodiments, the genetically engineered bacteria of the invention produce at least one detoxification molecule, anti- inflammation and/or gut barrier enhancer molecule in the presence of ROS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20- fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600- fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g. , by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays;

immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen).

[00339] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of an of a detoxification molecule, anti- inflammation and/or gut barrier enhancer molecule in the presence of ROS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the anti- inflammation and/or gut barrier enhancer molecule. In embodiments using genetically modified forms of these bacteria, the anti-inflammation and/or gut barrier enhancer molecule will be detectable in the presence of ROS.

[00340] In certain embodiments, the anti- inflammation and/or gut barrier enhancer molecule is butyrate. Methods of measuring butyrate levels, e.g. , by mass

spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g. , Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate

embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μΜ/OD, at least about 10 μΜ/OD, at least about 100 μΜ/OD, at least about 500 μΜ/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in the presence of ROS.

Constitutive promoters

[00341] In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.

[00342] In some embodiments, the constitutive promoter is active under in vivo conditions, e.g. , the gut, or in the presence of metabolites associated with certain diseases, such as toxicity-induced diarrhea or other toxicity- induced condition, as described herein. In some embodiments, the promoter is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut and/or in the presence of metabolites associated with certain diseases, such as toxicity-induced diarrhea or other toxicity-induced condition, as described herein, and under in vitro conditions, e.g. , various cell culture and/or cell production and/or manufacturing conditions, as described herein.

[00343] In some embodiments, the constitutive promoter that is operably linked to the gene encoding the payload is active in various exogenous environmental conditions {e.g. , in vivo and/or in vitro and/or production/manufacturing conditions). [00344] In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoter is active in low- oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the constitutive promoter is directly or indirectly induced by a molecule that is co- administered with the bacterial cell. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.

[00345] Bacterial constitutive promoters are known in the art. Examplary constitutive promoters are listed in the following Tables. The strength of the constitutive promoter can be further fine-tuned through the selection of ribosome binding sites of the desired strengths.

[00346] In some embodiments, the gene sequence(s) encoding a propionate catabolism enzyme is operably linked to a Escherichia coli σ70 promoter. Exemplary E. coli σ70 promoters are listed in Table 8.

[00347] In some embodiments, the gene sequence(s) encoding a detoxification molecule and/or anti-inflammation molecule and/or gut-barrier enhancing molecule is operably linked to a Escherichia coli σ70 promoter. Exemplary E. coli σ70 promoters are listed in Table 6A.

Table 8. Constitutive E. coli σ70 promoters

NO: 157 caggccggaataactccctataatgcgcca

SEQ ID constitutive promoter

BBa_J23100 35 NO: 158 family member ggctagctcagtcctaggtacagtgctagc

SEQ ID constitutive promoter

BBa_J23101 35 NO: 159 family member agctagctcagtcctaggtattatgctagc

SEQ ID constitutive promoter

BBa_J23102 35 NO: 160 family member agctagctcagtcctaggtactgtgctagc

SEQ ID constitutive promoter

BBa_J23103 35 NO: 161 family member agctagctcagtcctagggattatgctagc

SEQ ID constitutive promoter

BBa_J23104 35 NO: 162 family member agctagctcagtcctaggtattgtgctagc

SEQ ID constitutive promoter

BBa_J23105 35 NO: 163 family member ggctagctcagtcctaggtactatgctagc

SEQ ID constitutive promoter

BBa_J23106 35 NO: 164 family member ggctagctcagtcctaggtatagtgctagc

SEQ ID constitutive promoter

BBa_J23107 35 NO: 165 family member ggctagctcagccctaggtattatgctagc

SEQ ID constitutive promoter

BBa_J23108 35 NO: 166 family member agctagctcagtcctaggtataatgctagc

SEQ ID constitutive promoter

BBa_J23109 35 NO: 167 family member agctagctcagtcctagggactgtgctagc

SEQ ID constitutive promoter

BBa_J23110 35 NO: 168 family member ggctagctcagtcctaggtacaatgctagc

SEQ ID constitutive promoter

BBa_J23111 35 NO: 169 family member ggctagctcagtcctaggtatagtgctagc

SEQ ID constitutive promoter

BBa_J23112 35 NO: 170 family member agctagctcagtcctagggattatgctagc

SEQ ID constitutive promoter

BBa_J23113 35 NO: 171 family member ggctagctcagtcctagggattatgctagc

SEQ ID constitutive promoter

BBa_J23114 35 NO: 172 family member ggctagctcagtcctaggtacaatgctagc

SEQ ID constitutive promoter

BBa_J23115 35 NO: 173 family member agctagctcagcccttggtacaatgctagc

SEQ ID constitutive promoter

BBa_J23116 35 NO: 174 family member agctagctcagtcctagggactatgctagc

SEQ ID constitutive promoter

BBa_J23117 35 NO: 175 family member agctagctcagtcctagggattgtgctagc

SEQ ID constitutive promoter

BBa_J23118 35 NO: 176 family member ggctagctcagtcctaggtattgtgctagc

[00348] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to a E. coli σS promoters. Exemplary E. coli σS promoters are listed in Table 9.

Table 9. Constitutive E. coli σ promoters

[00349] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to a E. coli σ 32 promoters. Exemplary E. coli σ 32 promoters are listed in Table 10.

Table 10. Constitutive E. coli σ 32 promoters

[00350] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to a B. subtilis σ ^Α promoters. Exemplary B. subtilis σ ^Α promoters are listed in Table 11. Table 11. Constitutive B. subtilis σ ^Α promoters

[00351] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to a B. subtilis σΒ promoters. Exemplary B. subtilis σΒ promoters are listed in Table 12.

Table 12. Constitutive B. subtilis σ promoters

[00352] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to promoters from Salmonella. Exemplary Salmonella promoters are listed in Table 13. Table 13. Constitutive promoters from miscellaneous prokaryotes

[00353] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to promoters from bacteriophage T7. Exemplary promoters from bacteriophage T7 are listed in Table 14.

Table 14. Constitutive promoters from bacteriophage T7

[00354] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to promoters bacteriophage SP6. Exemplary promoters from bacteriophage SP6 are listed in Table 15.

Table 15. Constitutive promoters from bacteriophage SP6

[00355] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to promoters from yeast. Exemplary promoters from yeast are listed in Table 16. Table 16. Constitutive promoters from yeast

specific

[00356] In some embodiments, the gene sequence(s) encoding a detoxification molecule, ant i- inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to promoters from eukaryotes. Exemplary promoters from eukaryotes are listed in Table 17.

Table 17. Constitutive promoters from miscellaneous eukaryotes

[00357] Other exemplary promoters are listed in Table 18. Table 18. Other Constitutive Promoters

[00358] In some embodiments, the constitutive promoter is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID

NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID

NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID

NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID

NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID

NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID

NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID

NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID

NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID

NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID

NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID

NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID

NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID

NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID

NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID

NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID

NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID

NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID

NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID

NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID

NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID

NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID

NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID

NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID

NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID

NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID

NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID

NO: 297, and/or SEQ ID NO: 298.

Multiple Mechanisms of Action

[00359] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MO As), e.g. , circuits producing multiple copies of the same product (e.g. , to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene encoding a particular payload, e.g., a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene encoding a particular payload, e.g., a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, inserted at three different insertion sites and three copies of the gene encoding a different payload inserted at three different insertion sites. [00360] In some embodiments, the genetically engineered bacteria comprise one or more of (1) one or more gene(s) or gene cassette(s) for the production of propionate, as described herein (2) one or more gene(s) or gene cassette(s) for the production of butyrate, as described herein (3) one or more gene(s) or gene cassette(s) for the production of acetate, as described herein (4) one or more gene(s) or gene cassette(s) for the production of tryptophan and/or its metabolites (including but not limited to kynurenine, indole, indole acetic acid, indole-3 aldehyde, and IP A) , as described herein (5) one or more gene(s) or gene cassette(s) for the production of one or more of GLP-2 and GLP-2 analogs, as described herein (6) one or more gene(s) or gene cassette(s) for the production of human or viral or monommerized IL- 10, as described herein (7) one or more gene(s) or gene cassette(s) for the production of human IL- 22, as described herein (8) one or more gene(s) or gene cassette(s) for the production of IL-2, and./or SOD, and/or IL-27 and other interleukins, as described herein (9) one or more gene(s) or gene cassette(s) for the production of one or more transporters, e.g. for the import of tryptophan and/or metabolites as described herein (10) one or more polypepides for secretion, including but not limited to GLP-2 and its analogs, IL- 10, and/or IL-22, SCFA and/or tryptophan synthesis and/or catabolic enzymes in wild type or in mutated form (for increased stability or metabolic activity) (11) one or more components of secretion machinery, as described herein (12) one or more auxotrophies, e.g. , deltaThyA (13) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance (14) one or more mutations/deletions to increase the flux through a metabolic pathway encoded by one or more genes or gene cassette(s), e.g mutations/deletions in genes in NADH consuming pathways, genes involved in feedback inhibition of a metabolic pathway encoded by the gene(s) or gene cassette(s) genes, as described herein (15) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway, or (16) one or more gene(s) or gene cassette(s) encoding a detoxification molecule, e.g. , one or more enzymes capable of detoxifying a toxic or deleterious molecule.

[00361] In some embodiments, the genetically engineered bacteria promote one or more of the following effector functions: (1) neutralizes TNF-a, IFN-γ, IL- Ιβ, IL-6, IL-8, IL- 17, and/or chemokines, e.g., CXCL-8 and CCL2 (2) activates include AHR (e.g. , which result in IL-22 production) and (3) activates PXR, (4) inhibits HDACs, (5) activates GPR41 and/or GPR43 and/or GPR109A, (6) inhibits NF-kappaB signaling, (7) modulators of

PPARgamma, (8) activates of AMPK signaling, (9) modulates GLP- 1 secretion and/or (10). scavenges hydroxyl radicals and functions as antioxidants. [00362] In some embodiments, under conditions where the payload is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900- fold, at least about 1, 000-fold, or at least about 1, 500-fold more of the payload, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

[00363] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the payload gene(s). Primers specific for payload the gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35- 45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload gene(s).

[00364] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the payload gene(s). Primers specific for payload the gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35- 45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload gene(s).

[00365] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding short chain fatty acid production enzymes described herein and/or one or more gene sequence(s) encoding tryptophan catabolism enzyme(s) described herein and one or more gene sequence(s) encoding metabolite transporters described herein, and/or one or more gene sequence(s) encoding one or more therapeutic peptides for secretion, and/or one or more gene sequence(s) encoding a detoxification molecule, e.g. , one or more enzymes capable of detoxifying a toxic or deleterious molecule, as described herein.

[00366] In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette encoding a detoxification molecule, for example, an enzyme capable of detoxifying a toxic or deleterious molecule, as described herein. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate. In some embodiments, the genetically engineered bacteria comprise a propionate gene cassette and are capable of producing propionate. In some embodiments, the genetically engineered bacteria comprise a acetate gene cassette and are capable of producing acetate. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL- 10. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-2. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-22. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-27. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding GLP-2. In some embodiments, the genetically engineered bacteria are capable of producing kyurenine.

[00367] In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL- 10. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-2. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-22. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-27. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding SOD. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding GLP-2. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and are capable of producing kyurenine.

[00368] In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-10 and one or more gene sequences encoding IL-2, IL-22, IL-27, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine.

[00369] In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-2 and one or more gene sequences encoding IL-10, IL-22, IL-27, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-22 and one or more gene sequences encoding IL-2, IL-10, IL-27, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-27 and one or more gene sequences encoding IL-2, IL-22, IL-10, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding GLP-2 and one or more gene sequences encoding IL-2, IL-22, IL-27, IL-10, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine.

[00370] In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding SOD and one or more gene sequences encoding IL-2, IL-22, IL-27, GLP-2, and IL- 10. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine.

[00371] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-10 and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-22, IL-27, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-2 and a gene sequence(s) encoding one or more molecules selected from IL-10, IL-22, IL-27, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-22 and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-27, IL-10, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence(s) encoding IL-27 and a gene sequence encoding one or more molecules selected from IL-2, IL-22, IL-10, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding SOD and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-22, IL-27, GLP-2, and IL-10. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding GLP-2 and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-22, IL-27, IL-10, and SOD. In any of these embodiments, the genetically engineered bacteria are capable of producing kyurenine. In any of these embodiments, the genetically engineered bacteria are capable of producing butyrate. In any of these embodiments, the genetically engineered bacteria are capable of producing propionate. In any of these embodiments, the genetically engineered bacteria are capable of producing acetate.

[00372] In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a constitutive promoter. In another embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of an inducible promoter. In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a promoter that is directly or indirectly induced by inflammatory conditions. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g. , FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g. , can be exogenously added) in the gut, e.g., arabinose and tetracycline. Examples of inducible promoters include, but are not limited to, an FNR responsive promoter, a P _ara c promoter, a P _araBAD promoter, and a PietR promoter, each of which are described in more detail herein. Inducible promoters are described in more detail infra.

[00373] The at least one gene encoding the at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located on a plasmid in the bacterial cell. In another embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located in the chromosome of the bacterial cell, and at least one gene encoding at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion from a different species of bacteria are located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located on a plasmid in the bacterial cell, and at least one gene encoding the at least one one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion from a different species of bacteria are located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located in the chromosome of the bacterial cell, and at least one gene encoding the at least one one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion from a different species of bacteria are located in the chromosome of the bacterial cell.

[00374] In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed on a low-copy plasmid. In some embodiments, the gene

sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion.

[00375] In some embodiments, a recombinant bacterial cell of the invention comprising at least one gene encoding at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed on a high-copy plasmid do not increase tryptophan catabolism as compared to a recombinant bacterial cell comprising the same gene expressed on a low-copy plasmid in the absence of a heterologous importer of tryptophan and/or its metabolites and additional copies of a native importer of tryptophanand/or its metabolites. In alternate embodiments, the importer of tryptophan and/or its metabolites is used in conjunction with a high-copy plasmid.

Other Inducible Promoters

[00376] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an arabinose inducible system. The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub- saturating L-arabinose

concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L-arabinose transporter (Zhang et al., Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum; Appl. Environ.

Microbiol. August 2012 vol. 78 no. 16 5831-5838). Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

[00377] In one embodiment, expression of one or more protein(s) of interest, e.g., a detoxificatuion molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, is driven directly or indirectly by one or more arabinose inducible promoter(s).

[00378] In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some

embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., arabinose.

[00379] In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to

administration, e.g., arabinose. In some embodiments, the cultures, which are induced by arabinose, are grown arerobically. In some embodiments, the cultures, which are induced by arabinose, are grown anaerobically.

[00380] In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the arabinose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non- limiting example, the first inducing conditions may be culture conditions, e.g., including arabinose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more arabinose promoters drive expression of one or more protein(s) of interest, in combination with the FNR promoter driving the expression of the same gene sequence(s).

[00381] In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[00382] In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter

[00383] In one embodiment, the mmdA gene has at least about 80% identity with SEQ ID NO: 223. In another embodiment, the mmdA gene has at least about 85% identity with SEQ ID NO: 223. In one embodiment, the mmdA gene has at least about 90% identity with SEQ ID NO: 223. In one embodiment, the mmdA gene has at least about 95% identity with SEQ ID NO: 223. In another embodiment, the mmdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 223.. Accordingly, in one embodiment, the mmdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 223. In another embodiment, the mmdA gene comprises the sequence of SEQ ID NO: 223. In yet another embodiment the mmdA gene consists of the sequence of SEQ ID NO: 223.

[00384] In some embodiments, the genetically engineered bacteria comprise one or more methymalonyl-CoA epimerase (mmdA) gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences of SEQ ID NO: 67. In some

embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more methymalonyl-CoA epimerase gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences of SEQ ID NO: 66. In some embodiments, the genetically engineered bacteria comprise one or more methymalonyl-CoA epimerase gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 66.

[00385] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

[00386] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one

embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter.

[00387] In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some

embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., rhamnose

[00388] In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to

administration, e.g., rhamnose. In some embodiments, the cultures, which are induced by rhamnose, are grown arerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobic ally.

[00389] In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non- limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest and/or

transcriptional regulator(s), e.g., FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s).

[00390] In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences of SEQ ID NO: 25.

[00391] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-l- thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydro lyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacl) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by E. coli, its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate Lacl, can be used instead of IPTG in a similar manner.

[00392] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s).

[00393] In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., IPTG.

[00394] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some

embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to

administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown arerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically.

[00395] In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

[00396] In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[00397] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity with any of the sequences of SEQ ID NO:26. In some embodiments, the IPTG inducible construct further comprises a gene encoding lacl, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more acrB gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity with any of the sequences of SEQ ID NO: 75. In some

embodiments, the genetically engineered bacteria comprise one or more acrB gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 28.

[00398] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M & Bujard H.PNAS, 1992 Jun 15;89(12):5547-51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet- inducible promoter is reduced. Tetracycline-controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP 16 (virion protein 16) from herpes simplex virus. In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline- controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.

[00399] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a tetracycline inducible promoter. [00400] In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., tetracycline

[00401] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically.

[00402] In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the tetracycline inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., tetracycline and IPTG). In another non- limiting example, the first inducing conditions may be culture conditions, e.g., including tetracycline presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more tetracycline promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s). [00403] In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the

tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[00404] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the bolded sequences of SEQ ID NO: 334 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences of SEQ ID NO: 334 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 334 in italics (Tet repressor is in italics).

[00405] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive

mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et al., Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy; J Biotechnol. 2015 Jun 10; 203: 32-40, and references therein). Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage λ promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the λ promoters can then be efficiently regulated by the mutant thermo labile cI857 repressor of bacteriophage λ. At temperatures below 37 °C, cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in Fig. 22A. Inducible expression from the ParaBad can be controlled or further fine- tuned through the optimization of the ribosome binding site (RBS), as described herein.

[00406] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a thermoregulated promoter.

[00407] In some embodiments, the thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some

embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., temperature.

[00408] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shup off production of the one or more protein(s) of interest. This can be done in a thermoregulated system by growing the strain at lower temperatures, e.g., 30 C. Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, the thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically.

[00409] In one embodiment, the thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during

preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabinose). In another non- limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

[00410] In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[00411] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences of SEQ ID NO: 332. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 333. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 333.

[00412] The nucleic acid sequence a gene encoding mutant cI857 repressor is provided below:

TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACT CTC ATTGCATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATC CCT GATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAATCACCTGG CTC AACAGCCTGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGGCTTGGAGCC TGT TGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTT GGT TGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTTAGGTGAGAACAT CCC TGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGGCTGCATACT AAC CGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAAATTCTTCAACGCTAAC TTT GAGAATTTTTGTAAGCAATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAA AGC ACCAACGCCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGCCAAG TTC ATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGCTCTTGTGT TAA TGGTTTCTTTTTTGTGCTCAT (SEQ ID NO: 332) [00413] The amino acid sequence a mutant cI857 repressor is provided below:

MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQSGVGALFNGINA LNAYNA ALLTKILKVSVEEFSPS IAREIYEMYEAVSMQPSLRSEYEYPVFSHVQAGMFSPKLRTFTKGD AERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGMLILVDPEQAVEPGDFCIARLG GDE FTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSVVGKVIASQWPEETFG (SEQ ID NO: 333)

[00414] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.

[00415] This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control.

[00416] In one embodiment, expression of one or more protein(s) of interest is indirectly regulated by a repressor expressed under the control of one or more PssB

promoter(s).

[00417] In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest .In some embodiments, induction of the RssB promoter(s) indirectly drives the expression of one or more protein(s) of interest during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, conditions for induction of the RssB promoter(s) are provided in culture, e.g., in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.

[00418] In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[00419] In another non- limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic conditions, e.g., the gut, but prevent survival under aerobic conditions

(bio safety switch).

[00420] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the sequences described herein.

Induction of Payloads During Strain Culture

[00421] In some embodiments, it is desirable to pre-induce the expression and/or activity of the payload or protein of interest, e.g., detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, prior to administration.

[00422] In some embodiments, the payload is capable of detoxifying a deleterious molecule. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term "bacterial culture" or bacterial cell culture" or "culture" refers to bacterial cells or microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term "fermentation" refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like. [00423] In some embodiments, the payload is carboxypeptidase Gi (CPD Gi) or carboxypeptidase G ₂ (CPD G ₂ ). In some embodiments, the payload is D-saccharic acid 1, 4- lactone (SAL). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen.. In some embodiments, the payload is a proton pump inhibitor. In some embodiments, the payload is a heavy metal chelator. In some embodiments, the payload is a plant phytochelatin. In some embodiments, the payload is a short-chained fatty acid, e.g. butyrate, propionate, or acetate. In some embodiments, the payload is the enzyme Pseudomonas.

[00424] Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium. In some

embodiments, phenylalanine is added to the media, e.g., to boost cell health. Without wishing to be bound by theory, addition of phenylalanine to the medium may prevent bacteria from catabolizing endogenously produced phenylalanine required for cell growth.

[00425] In some embodiments, the one or more protein(s) of interest and are directly or indirectly induced, while the strains is grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is "wasted", in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut (e.g., low oxygen, or in the presence of gut metabolites).

[00426] In one embodiment, expression of one or more payload(s), is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s) is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s) is driven from the one or more different promoters.

[00427] In some embodiments, the strains are administered without any pre- induction protocols during strain growth prior to in vivo administration.

Anaerobic induction

[00428] In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10 ^{^} 8 to 1X10 ^{^} 11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s) under the control of one or more FNR promoters.

[00429] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions.

[00430] In one embodiment, expression of two or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more Payload(s under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under anaerobic or low oxygen conditions.

[00431] Without wishing to be bound by theory, strains that comprise one or more payload(s) and/or transcriptional regulator(s) under the control of an FNR promoter, may allow expression of payload(s) and/or transcriptional regulator(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut.

[00432] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some

embodiments, strains may comprise one or more payload gene sequence(s) and/or under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

[00433] In one embodiment, expression of one or more payload is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[00434] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions.

[00435] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g. , FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions. Aerobic induction

[00436] In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g. , for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g. , ranging from 1X10 ^{^} 8 to 1X10 ^{^} 11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours.

[00437] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

[00438] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions.

[00439] In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[00440] In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

[00441] In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more

thermoregulated promoter(s)and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions.

[00442] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions.

[00443] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one

embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[00444] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under

thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under aerobic conditions.

[00445] In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[00446] In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[00447] In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some

embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[00448] In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene payload for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[00449] As evident from the above non- limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with thermoregulation. Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions.

Microaerobic Induction

[00450] In some embodiments, viability, growth, and activity are optimized by pre-inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to "strike a balance" between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s) are driven by a anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10 ^{^} 8 to 1X10 ^{^} 11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for

approximately 3 to 5 hours.

[00451] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

[00452] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions.

[00453] Without wishing to be bound by theory, strains that comprise one or more payload(s) and/or transcriptional regulator(s) under the control of an FNR promoter, may allow expression of payload(s) and/or transcriptional regulator(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut.

[00454] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

[00455] In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

[00456] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions.

[00457] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g. , an anaerobic/low oxygen promoter or microaerobic promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Induction of Strains using Phasing, Pulsing and/or Cycling

[00458] In some embodiments, cycling, phasing, or pulsing techniques are emplyed during cell growth, expansion, recovery, purification, fermentation, and/or

manufacture to efficienty induce and grow the strains prior to in vivo administration. This method is used to "strike a balance" between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g. , for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g. , ODs within

8 11 the range of 0.1 to 10, indicating a certain density e.g. , ranging from 1X10 to 1X10 ,and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g. , reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle.

[00459] In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non- liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g. , induction of FNR promoters). In one

embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed.

[00460] In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non- limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g. , for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1X10 to 1X10 ¹¹ . Then the chemical inducer, e.g. , arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed.

[00461] In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload. For example, lowering the temperature during culture may improve the proper folding of the payload. In such instances, cells are first grown at a temperature optimal for growth (e.g., 37 C). In some embodiments, the cells are then induced, e.g. , by a chemical inducer, to express the payload. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload, e.g., a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule.

[00462] In some embodiments, payload(s) are under the control of different inducible promoters, for example two different chemical inducers. In other embodiments, the payload is induced under low oxygen conditions or microaerobic conditions and a second payload is induced by a chemical inducer.

[00463] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques.

[00464] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques.

[00465] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some

embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) and /or

transcriptional regulator gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein, and are induced through the employment of phasing or cycling or pulsing or spiking techniques.

[00466] Any of the strains described herein can be grown through the

employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions.

[00467] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques.

[00468] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. Any of the strains described in these embodiments may be induced through the employment of phasing or cycling or pulsing or spiking techniques.

Aerobic induction of the FNR promoter

[00469] FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, oxygen bypass system shown and described in Fig. 19A is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression a protein of interest by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of a protein of interest. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of a protein of interest.

[00470] In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non- limiting example, a protein of interest and FNRS24Y can in some embodiments be induced simultaneously, e.g. , from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter.

[00471] In some embodiments, a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

[00472] Sequences useful for expression from inducible promoters are listed in

Table 8.

Table 8. Inducible promoter construct sequences

Secretion

[00473] In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting a molecule from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.

[00474] In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane- spanning secretion system. Membrane- spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; CoUinson et al., 2015; Costa et al, 2015; Reeves et al, 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in FIG. 37, FIG. 38, FIG. 39, FIG. 40, and FIG. 41. Mycobacteria, which have a Gram- negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al, 2003). With the exception of the T2SS, double membrane- spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane- spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane- spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).

[00475] In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type Ill-like secretion system (T3SS) from Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologouse protein or peptide comprises a type III secretion sequence that allows the molecule of interest o be secreted from the bacteria. [00476] In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173- bp region, is fused to the gene encoding the polypeptide of interest can be used to secrete heterologous polypeptides (See, e.g., Majander et al., Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol. 2005

Apr;23(4):475-81). In some cases, the untranslated region from the fliC loci, may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g., Duan et al, Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 23 7437- 7438).

[00477] In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in FIG. 38, a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta- domain of an autotransporter. The N-terminal, Sec-dependent signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex ('Beta-barrel assembly machinery') where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane-associated peptidase (black scissors; right side of Bam complex) to a

complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.

[00478] In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, , e.g., a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. FIG. 39 shows the alpha- hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP- binding cassette transporter; HlyD, a membrane fusion protein; and TolC , an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[00479] In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane- spanning secretion system. Single membrane- spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in E. coli), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis,

Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii,

Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads. [00480] In order to translocate a protein of interest, e.g., a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g. , therapeutic polypeptides) - particularly those of eukaryotic origin - contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

[00481] One way to secrete properly folded proteins in gram- negative bacteria- particularly those requiring disulphide bonds - is to target the reducing-environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These "leaky" gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a "leaky" or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be

accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at -500,000 copies per cell and functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. 1. Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). TolA-PAL and OmpA

complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpl can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes, in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[00482] To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from lpp, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl, under the control of an inducible promoter. For example, expression of lpp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., over expression of colicins or the third topological domain of TolA, which peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

[00483] The Table 9 and Table 10 below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

Table 9 Secretion systems for gram positive bacteria

Table 10. Secretion Systems for Gram negative bacteria

[00484] The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe / Volume 1, Number 9, 2006 "Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently." the contents of which is herein incorporated by reference in its entirety.

[00485] Any of the secretion systems described herein may according to the disclosure be employed to secrete the proteins of interest. Non-limiting examples of proteins of interest include e.g. , carboxypeptidase Gi (CPD Gi), carboxypeptidase G ₂ (CPD G ₂ ), D- saccharic acid 1, 4-lactone (SAL), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, proton pump inhibitor, heavy metal chelator, plant phytochelatin, GLP-2 peptides, GLP-2 analogs, IL-22, vIL- 10, hIL- 10, monomerized IL- 10, IL-27, IL- 19, IL-20, IL- 24, tryptophan synthesies enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, including but not limited to IDO, TDO, kynureninase, other tryptophan pathway catabolic enzymes, e.g. in the indole pathway and/or the kynurenine pathway as described herein. These polypeptides may be mutated to increase stability, resistance to protease digestion, and/or activity.

Table 11. Comparison of Secretion systems for secretion of polypeptide from engineered bacteria

[00486] In some embodiments, the therapeutic polypeptides of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a therapeutic polypeptide of interest, e.g. , carboxypeptidase Gi (CPD Gi),

carboxypeptidase G ₂ (CPD G ₂ ), D-saccharic acid 1, 4-lactone (SAL), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN- 38), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, proton pump inhibitor, heavy metal chelator, plant phytochelatin,GLP-2 peptides, GLP-2 analogs, IL-22, vIL- 10, hIL- 10, monomerized IL- 10, IL-27, IL- 19, IL-20, IL-24, is assembled behind a fliC-5'UTR (e.g. , 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the expression of the therapeutic peptide of interested secreted using components of the flagellar type III secretion system is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g. , FNR- inducible promoter), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g. , can be exogenously added) in the gut, e.g., arabinose is used. In some embodiments, the therapeutic polypeptide of interest is expressed from a plasmid (e.g. , a medium copy plasmid). In some embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g. , the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency.

[00487] In some embodiments, the therapeutic polypeptides of interest, e.g., a detoxification molecule, e.g. , carboxypeptidase Gi (CPD Gi), carboxypeptidase G ₂ (CPD G ₂ ), D-saccharic acid 1, 4-lactone (SAL), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, proton pump inhibitor, heavy metal chelator, plant phytochelatin; an antiinflammatory molecule; and/or a gut barrier enhancer molecule, e.g., GLP-2 peptides, GLP-2 analogs, IL-22, vIL- 10, hIL- 10, monomerized IL- 10, IL-27, IL- 19, IL-20, IL-24, are secreted using via a diffusible outer membrane (DOM) system. In some embodiments, the therapeutic polypeptide of interest is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, the therapeutic polypeptide of interest is fused to a Tat- dependent secretion signal. Exemplary Tat-dependent tags include TorA, FdnG, and DmsA. In some embodiments, expression of the secretion-tagged therapeutic protein is driven by a tet promoter or an inducible promoter, such as oxygen level-dependent promoters (e.g. , FNR- inducible promoter), or by promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. In some embodiments, the secretion-tagged therapeutic polypeptide of interest is expressed from a plasmid (e.g. , a medium copy plasmid). In other embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into the bacterial chromosome, e.g., at one or more of the integration sites shown in FIG. 24. In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include lpp, pal, tolA, and/or nlpl. In some embodiments, lpp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, tolA is deleted or mutated. In other embodiments, nlpl is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g. , to increase stability of the polypeptide in the periplasm. Non- limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated.

[00488] In some embodiments, the therapeutic polypeptides of interest, e.g., an enzyme capable of detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), carboxypeptidase G ₂ (CPD G ₂ ), D-saccharic acid 1, 4-lactone (SAL), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN- 38), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, proton pump inhibitor, heavy metal chelator, plant phytochelatin; an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule, e.g. , GLP-2 peptides, GLP-2 analogs, IL-22, vIL- 10, hIL- 10, monomerized IL- 10, IL-27, IL- 19, IL-20, IL-24, are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodimetns, the therapeutic protein of interest is expressed as a fusion protein with the native Nissle auto-secreter E. coli_01635 (where the original passenger protein is replaced with the therapeutic polypeptides of interest.

[00489] In some embodiments, the therapeutic polypeptides of interest, e.g. , e.g., carboxypeptidase Gi (CPD Gi), carboxypeptidase G ₂ (CPD G ₂ ), D-saccharic acid 1, 4-lactone (SAL), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10- hydroxycamptothecin (SN-38), molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, proton pump inhibitor, heavy metal chelator, plant phytochelatin, GLP-2 peptides, GLP-2 analogs, IL-22, vIL- 10, hIL- 10, monomerized IL- 10, IL-27, IL- 19, IL-20, IL-24, are secreted via Type I Hemolysin Secretion. In one embodiment, therapeutic polypeptide of interest is expressed as fusion protein with the 53 amino acids of the C terminus of alpha-hemolysin (hlyA) of E. coli CFT073.

Essential genes and auxotrophs

[00490] As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference). [00491] An "essential gene" may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the genetically engineered bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.

[00492] An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil, as long as the corresponding wild-type gene product is not produced in the bacteria.

[00493] Table 12 lists exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.

Table 12. Non-limiting Examples of Bacterial Genes Useful for Generation of an Auxotroph

[00494] Table 13 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.

Table 13. Survival of amino acid auxotrophs in the mouse gut

[00495] For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al, 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the

auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[00496] Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g. , by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[00497] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g. , by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g. , outside of the gut).

[00498] In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non- auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

[00499] Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gap A, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zip A, dapE, dap A, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, sec A, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsi, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, lnt, glnS, lldA, cydA, A, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fab A, mviN, rne, yceQ, fabD, fabG, acpP, tmk, ho IB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, top A, rib A, fabl, racR, die A, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

[00500] In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson "Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, "ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

[00501] In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG, and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G.

[00502] In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3- acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2- aminobenzothiazole, indole- 3 -butyric acid, indole- 3 -acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2- aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

[00503] In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).

[00504] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.

[00505] In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill- switch components and systems described herein. For example, the genetically engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al, "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill- switch circuitry, such as any of the kill- switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (Wright et ah, 2015). In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that produce the enzyme capable of detoxifying a deleterious molecule, antiinflammatory, or gut barrier enhancer molecule.

[00506] In one embodiment, a genetically engineered bacterium, comprises one or more biosafety constructs integrated into the bacterial chromosome in combination with one or more biosafety plasmid(s). In some embodiments, the plasmid comprises a conditional origin of replication (COR), for which the plasmid replication initiator protein is provided in trans, i.e., is encoded by the chromosomally integrated biosafety construct. In some embodiments, the chromosomally integrated construct is further introduced into the host such that an auxotrophy results (e.g., dapA or thyA auxotrophy), which in turn is complemented by a gene product expressed from the biosafety plasmid construct. In some embodiments, the biosafety plasmid further encodes a broad- spectrum toxin (e.g., Kis), while the integrated biosafety construct encodes an anti-toxin (e.g., anti-Kis), permitting propagation of the plasmid in the bacterial cell containing both constructs. Without wishing to be bound by theory, this mechanism functions to select against plasmid spread by making the plasmid DNA itself disadvantageous to maintain by a wild-type bacterium. A non-limiting example of such a biosafety system is shown in Fig. 36A, Fig. 36B, Fig. 36C, and Fig. 36D.

[00507] In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that produce the payload and/or reduce the deleterious molecule. In a more specific aspect, auxotrophic modifications may be used to screen for mutant bacteria that consume inhibit and/or metabolize the deleterious molecule.

Genetic regulatory circuits

[00508] In some embodiments, the genetically engineered bacteria comprise multi-layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811 and PCT/US2016/39434, both of which are incorporated herein by reference in its entirety). The genetic regulatory circuits are useful to screen for mutant bacteria that inhibit or metabolize a deleterious molecule or rescue an auxotroph. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.

[00509] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the payload is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the payload is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

[00510] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf- lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the tetR, and the payload is expressed.

[00511] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.

[00512] Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

[00513] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a payload. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the payload. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing payload translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR- responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.

[00514] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR- responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed.

[00515] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a payload operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the payload remains in the 3' to 5' orientation, and no functional payload is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the payload is reverted to the 5' to 3' orientation, and functional payload is produced.

[00516] In some embodiments, the invention provides genetically engineered bacteria comprising a payload gene or gene cassette and a polymerase- and recombinase- regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR- responsive promoter; a second gene or gene cassette for producing a payload operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload. The third gene encoding the T7 polymerase is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3' to 5' orientation, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5' to 3' orientation, and the payload is expressed.

[00517] Synthetic gene circuits expressed on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a therapeutic molecule and further comprise a toxin-anti-toxin system that simultaneously produces a toxin (hok) and a short-lived anti-toxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015). In some embodiments, the genetically engineered bacteria further comprise alp7 from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015).

Host-plasmid mutual dependency

[00518] In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad- spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin {e.g., a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention.

[00519] The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxo trophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

[00520] Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of detoxifying deleterious molecules and further comprise a toxin-antitoxin system that simultaneously produces a toxin (hok) and a short-lived antitoxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015). In some embodiments, the genetically engineered bacteria further comprise alp7 from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015).

Kill switch

[00521] In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329, and 62/277,654, each of which is incorporated herein by reference in their entireties). The kill switch is intended to actively kill engineered microbes in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

[00522] Bacteria comprising kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a bio fuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to modulate or treat a disorder or condition may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a therapeutic payload gene, or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of the payload, , e.g., detoxification molecule, ant i- inflammatory molecule, and/or gut barrier enhancer molecule. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of the payload, e.g., detoxification molecule, ant i- inflammatory molecule, and/or gut barrier enhancer molecule. Alternatively, the bacteria may be engineered to die after the bacterium has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the

microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject).

Examples of such toxins that can be used in kill- switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation

(riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND

riboregulator switch is activated by tetracycline, isopropyl β-D-l-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al, 2010).

[00523] Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal {e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.

[00524] Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low-oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then

constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable.

[00525] In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one

recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[00526] In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[00527] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.

[00528] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.

[00529] In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase. [00530] In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

[00531] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.

[00532] In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxbl, PhiC31, TP901, Bxbl, PhiC31, TP901, HK022, HP1, R4, Intl, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, IntlO, Intl l, Intl2, Intl3, Intl4, Intl5, Intl6, Intl7, Intl8, Intl9, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.

[00533] In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill- switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) is shown in the figures. The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the AraBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxing gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arbinoase system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

[00534] Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an antitoxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

[00535] Arabinose inducible promoters are known in the art, including P _ara , P _ara B,

P _araC , and P _araBAD - In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the P _ara c promoter and the P _araBAD promoter operate as a bidirectional promoter, with the P _araBAD promoter controlling expression of a heterologous gene(s) in one direction, and the P _ara c (in close proximity to, and on the opposite strand from the P _araBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

[00536] In one exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P _araBAD promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a P _ara c promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (P _TetR ). In the presence of arabinose, the AraC transcription factor activates the P _araBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the the P _araBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the AraC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore

constitutively expressed. [00537] In one embodiment of the disclosure, the genetically engineered bacterium further comprises an antitoxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the antitoxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the antitoxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.

[00538] In another embodiment of the disclosure, the genetically engineered bacterium further comprises an antitoxin under the control of the P _araBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.

[00539] In another exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P _araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a P _ara c promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the P _araBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the the P _araBAD promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of P _araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill- switch system described directly above. In some embodiments, the sequence of P _araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anto-toxin kill- switch system described directly above. [00540] In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer- lived toxin killing it.

[00541] In some embodiments, the engineered bacteria of the present disclosure further comprise the gene(s) encoding the components of any of the above-described kill- switch circuits.

[00542] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, lbs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hip A, microcin B, microcin B 17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin lb, colicin 5, colicinlO, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6, colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

[00543] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNAl, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccE ^CTD , MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, ImlO, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

[00544] In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.

[00545] In some embodiments, the engineered bacteria provided herein express a payload that is controlled by a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from the fumarate and nitrate reductase regulator (FNR) promoter, arginine deiminiase and nitrate reduction (ANR) promoter, and dissimilatory nitrate respiration regulator (DNR) promoter.

[00546] In some embodiments, the genetically engineered bacterium provided herein is an auxotroph. In one embodiment, the genetically engineered bacterium is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thy A, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[00547] In some embodiments, the genetically engineered bacterium provided herein further comprises a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin.

[00548] In some embodiments, the genetically engineered bacterium is an auxotroph comprising a therapeutic payload, e.g., a detoxification molecule, ant i- inflammatory molecule, and/or gut barrier enhancer molecule, and further comprises a kill- switch circuit, such as any of the kill-switch circuits described herein.

[00549] In some embodiments of the above described genetically engineered bacteria, the gene(s) encoding the payload is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene(s) encoding the payload is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[00550] In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette for producing the edetoxification molecule, anti- inflammation and/or gut barrier enhancer molecule is present on a plasmid in the bacterium and operatively linked on the plasmid to the inducible promoter. In other embodiments, the gene or gene cassette for producing the detoxification molecule, anti- inflammation and/or gut barrier enhancermolecule is present in the bacterial chromosome and is operatively linked in the chromosome to the inducible promoter.

Methods of Screening

Mutagenesis

[00551] In some embodiments, the inducible promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, the inducible promoter is mutagenized, and mutants are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence-activated cell sorting (FACS) when the detectable product fluoresces. In some embodiments, one or more transcription factor binding sites is mutagenized to increase or decrease binding. In alternate embodiments, the wild-type binding sites are left intact and the remainder of the regulatory region is subjected to mutagenesis. In some embodiments, the mutant promoter is inserted into the genetically engineered bacteria of the invention to increase expression of the detoxification molecule, anti- inflammation molecule, and/or gut barrier enhancer molecule under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the inducible promoter and/or corresponding transcription factor is a synthetic, non-naturally occurring sequence.

[00552] In some embodiments, the gene encoding the detoxification molecule, anti- inflammation molecule, and/or gut barrier enhancer molecule is mutated to increase expression and/or stability of said molecule under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, one or more of the genes in a gene cassette for producing the detoxification molecule, anti- inflammation molecule, and/or gut barrier enhancer molecule is mutated to increase expression of said molecule under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the efficacy or activity of any of the importers and exporters for metabolites of interest can be improved through mutations in any of these genes. Mutations increase uptake or export of such metabolites, including but not limited to, tryptophan, e.g. , under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. Methods for directed mutation and screening are known in the art. Generation of Bacterial Strains with Enhance Ability to Transport Metabolites of

Interest

[00553] Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

[00554] This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite.

[00555] For example, if the biosynthetic pathway for producing a metabolite of interest is disrupted a strain capable of high-affinity capture of the metabolite of interest can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic metabolite of interest, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the metabolite of interest at regular intervals. Over time, cells that are most competitive for the metabolite of interest - at growth-limiting concentrations - will come to dominate the population. These strains will likely have mutations in their metabolite of interest-transporters resulting in increased ability to import the essential and limiting metabolite of interest.

[00556] Similarly, by using an auxotroph that cannot use an upstream metabolite to form the metabolite of interest, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite of interest. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways.

[00557] A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate.

[00558] Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth- limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

[00559] Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

[00560] Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations "screened" throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 10 11 2 CCD 1. This rate can be accelerated by the addition of chemical mutagens to the cultures - such as N-methyl-N-nitro-N- nitrosoguanidine (NTG) - which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted. [00561] At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvo luted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. 0. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

[00562] Similar methods can be used to generate E. coli Nissle mutants that consume or import metabolites, including, but not limited to, tryptophan.

Pharmaceutical compositions and formulations

[00563] Pharmaceutical compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent a disorder or a condition caused by a toxic molecule, metabolite, or other deleterious molecule.

Pharmaceutical compositions comprising the genetically engineered microorganisms of the invention may also be used to inhibit inflammatory mechanisms in the gut, restore and tighten gut mucosal barrier function, and/or treat or prevent autoimmunedisorders. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[00564] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein. In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein, e.g., to produce a detoxification molecule, anti- inflammation molecule, and/or gut barrier enhancer molecule. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein. [00565] The pharmaceutical compositions of the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into

compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of

administration.

[00566] The genetically engineered bacteria of the invention may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g. , oral, topical, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10 ⁵ to 10 ¹² bacteria, e.g., approximately 10 ⁵ bacteria, approximately 10 ⁶ bacteria,

7 8 9

approximately 10 bacteria, approximately 10 bacteria, approximately 10 bacteria, approximately 10 ¹⁰ bacteria, approximately 10 ¹¹ bacteria, or approximately 10 ¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In one embodiment, the

pharmaceutical composition is administered before the subject eats a meal. In one

embodiment, the pharmaceutical composition is administered currently with a meal. In on embodiment, the pharmaceutical composition is administered after the subject eats a meal.

[00567] The genetically engineered bacteria may be formulated into

pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g. , 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[00568] The genetically engineered microorganisms may be administered intravenously, e.g. , by infusion or injection.

[00569] The genetically engineered microroganisms of the disclosure may be administered intrathecally. In some embodiments, the genetically engineered microorganisms of the invention may be administered orally.

[00570] The genetically engineered bacteria of the invention may be

administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g. , preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g. , osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g. , a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.

[00571] The genetically engineered bacteria of the invention may be

administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium

carbomethylcellulose; and/or physiologically acceptable polymers such as

polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[00572] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. , pregelatinised maize starch, polyvinylpyrrolidone, hydro xypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g. , sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate- polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydro ymethylacry late- methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA- MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N- dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch

polymethacrylates, polyamino acids, and enteric coating polymers.

[00573] In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the small or large intestines. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

[00574] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g. , sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g. , methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria of the invention.

[00575] In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

[00576] In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, "flavor" is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

[00577] In certain embodiments, the genetically engineered bacteria of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[00578] In another embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria- fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. For example, see U.S. 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.

[00579] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal

administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g. , conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[00580] The genetically engineered bacteria of the invention may be

administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoro methane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).

Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g. , of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[00581] The genetically engineered bacteria of the invention may be

administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g. , as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[00582] In some embodiments, the invention provides pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g. , single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g. , by infusion.

[00583] Single dosage forms of the pharmaceutical composition of the invention may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[00584] Dosage regimens may be adjusted to provide a therapeutic response.

Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index.

Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

[00585] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g. , U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly( methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly( vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and

polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[00586] The genetically engineered bacteria of the invention may be

administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

[00587] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. [00588] The pharmaceutical compositions of the invention may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions of the invention is supplied as a dry sterilized lyophilized powder or water- free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the

prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0- 10% sucrose (optimally 0.5- 1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a

concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g. , hyaluronidase.

[00589] Dosing can depend on several factors, including severity and

responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD ₅₀ , ED ₅₀ , EC ₅₀ , and IC ₅₀ may be determined, and the dose ratio between toxic and therapeutic effects (LD ₅₀ /ED ₅₀ ) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

Methods of treatment

[00590] Another aspect of the invention provides methods of modulating or treating a disorder or condition caused by a toxic molecule, metabolite, or other deleterious molecule. Another aspect of the invention provides methods of treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other conditios that may result from exposure to a toxic molecule, e.g., a chemotherapy- induced diarrhea and/or intestinal damage and that benefit from reduced gut inflammation and/or enhanced gut barrier function. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these disorders or conditions. In some embodiments, the subject to be treated is a human patient. In alternate embodiments, the subject to be treated is a veterinary subject.

[00591] In some embodiments, the disorder or condition is heavy metal poisoning, aluminum poisoning, antimony poisoning, arsenic poisoning, barium poisoning, bismuth poisoning, cadmium poisoning, chromium poisoning, cobalt poisoning, copper poisoning, gold poisoning, iron poisoning, lead poisoning, lithium poisoning, manganese poisoning, mercury poisoning, nickel poisoning, phosphorous poisoning, platinum poisoning, selenium poisoning, silver poisoning, thallium poisoning, tin poisoning, and zinc poisoning. In some embodiments, the genetically engineered bacteria and pharmaceutical compositions described herein are capable of sequestering the heavy metal, thereby reducing toxicity.

[00592] In some embodiments, the disorder or condition is chemotherapy- induced diarrhea and/or intestinal damage. Non- limiting examples include panenteritis, enterocolitis, mucositis, and/or diarrhea caused by antimetabolites, methotrexate, cytosine arabinoside, fluoropyrimidines, fluorouracil, capecitabine, tegafur-uracil, multitargeted folinic acid antagonists, pemetrexed, raltitrexed, gemcitabine, plant alkaloids, vinca alkaloids, vincristine, vinorelbine, epipodophyllotoxins, etoposide, taxanes, paclitaxel, docetaxel, topoisomerase I inhibitors, irinotecan, cytotoxic antibiotics, anthracyclines, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, alkylating agents, cyclophosphamide, platinums, cisplatin, carboplatin, oxaliplatin, and nedaplatin; abdominal pain and/or diarrhea caused by gemcitabine; autoimmune colitis and/or diarrhea caused by antibodies such as ipilumumab; ischemic colitis and/or diarrhea caused by plant alkaloids, taxanes, paclitaxel, docetaxel, and antibodies against VEGF such as bevacizumab; and/or metabolites or byproducts of those drugs (Andreyev et al., 2014). In some embodiments, the genetically engineered bacteria and pharmaceutical compositions described herein are capable of reducing the chemotherapy-induced intestinal damage and/or diarrhea, thereby reducing dose-limitations for the chemotherapeutic drug. In certain embodiments, the genetically engineered bacteria and pharmaceutical compositions described herein are capable of grade 1 diarrhea, grade 2 diarrhea, grade 3 diarrhea, grade 4 diarrhea, and/or grade 5 diarrhea. In some embodiments, the genetically engineered bacteria and pharmaceutical compositions described herein are capable of treating one or more symptoms of grade 1 diarrhea, grade 2 diarrhea, grade 3 diarrhea, grade 4 diarrhea, and/or grade 5 diarrhea, e.g., cramping.

[00593] In some embodiments, the disorder or condition is NSAID-induced diarrhea and/or intestinal damage. Non-limiting examples include naproxen-, indomethacin-, ketoprofen-, piroxicam-, ibuprofen-, diclofenac-, and COX-2 inhibitor- induced diarrhea and/or intestinal damage. In some embodiments, the genetically engineered bacteria and

pharmaceutical compositions described herein are capable of reducing the NSAID-induced intestinal damage and/or diarrhea, thereby reducing dose-limitations for the NSAID.

[00594] In some embodiments, the diarrheal disease is selected from the group consisting of acute watery diarrhea, e.g., cholera, acute bloody diarrhea, e.g. , dysentery, and persistent diarrhea. In some embodiments, the IBD or related disease is selected from the group consisting of Crohn' s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, intermediate colitis, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis. In some embodiments, the disease or condition is an autoimmune disorder selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospho lipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osto myelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn' s disease, Cogan' s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus,

Dressier' s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to diarrhea, bloody stool, mouth sores, perianal disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron deficiency, anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal development, and inflammation of the skin, eyes, joints, liver, and bile ducts. In some embodiments, the invention provides methods for reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing a systemic autoimmune disorder, e.g., asthma (Arrieta et al., 2015). [00595] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally, e.g. , in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g. , by enema. In some embodiments, the genetically engineered bacteria of the invention are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically.

[00596] In some embodiments, the genetically engineered viruses are prepared for delivery, taking into consideration the need for efficient delivery and for overcoming the host antiviral immune response. Approaches to evade antiviral response include the

administration of different viral serotypes as par of the treatment regimen (serotype switching), formulation, such as polymer coating to mask the virus from antibody recognition and the use of cells as delivery vehicles.

[00597] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g. , U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly( methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly( vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and

polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[00598] The genetically engineered bacteria of the invention may be

administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

[00599] In certain embodiments, administering the pharmaceutical composition to the subject reduces concentrations of the toxic molecule, metabolite, or other deleterious molecule in a subject. In some embodiments, the methods of the present disclosure may reduce the concentration of the toxic molecule, metabolite, or other deleterious molecule in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing the concentration of the toxic molecule, metabolite, or other deleterious molecule in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating the disorder or condition allows one or more symptoms of the disorder or condition to improve by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[00600] Before, during, and after the administration of the pharmaceutical composition, concentrations of the toxic molecule, metabolite, other deleterious molecule, or byproduct in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the invention to reduce toxic molecule, metabolite, or other deleterious molecule concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's toxic molecule, metabolite, or other deleterious molecule concentrations prior to treatment.

[00601] In some embodiments, reduction is measured by comparing the levels of inflammation in a subject before and after administration of the pharmaceutical composition. In one embodiment, the levels of inflammation is reduced in the gut of the subject. In one embodiment, gut barrier function is enhanced in the gut of the subject. In another embodiment, levels of inflammation is reduced in the blood of the subject. In another embodiment, the levels of inflammation is reduced in the plasma of the subject. In another embodiment, levels of inflammation is reduced in the brain of the subject.

[00602] In one embodiment, the pharmaceutical composition described herein is administered to reduce levels of inflammation in a subject to normal levels. In another embodiment, the pharmaceutical composition described herein is administered to reduce levels of inflammation in a subject below normal.

[00603] In some embodiments, the method of treating the disorder allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of treating the disorder, allows one or more symptoms of the condition or disorder to improve by at least about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold.

[00604] Before, during, and after the administration of the pharmaceutical composition, gut inflammation and/or barrier function in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the invention to enhance gut barrier function and/or to reduce gut inflammation to baseline levels, e.g., levels comparable to those of a healthy control, in a subject. In some embodiments, the methods may include administration of the compositions of the invention to reduce gut inflammation to undetectable levels in a subject, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's levels prior to treatment. In some embodiments, the methods may include administration of the compositions of the invention to enhance gut barrier function in a subject by about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100% or more of the subject's levels prior to treatment.

[00605] In certain embodiments, the genetically engineered bacterium

comprising the therapeutic payload is E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009), or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the gene or gene cassette for producing the payload may be re- administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice is shown in Fig. 26 In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

[00606] The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, including but not limited to, sodium phenylbutyrate, sodium benzoate, and glycerol phenylbutyrate. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria. In some embodiments, the pharmaceutical composition is administered with the chemo therapeutic. In alternate embodiments, the pharmaceutical composition is administered before or after administering the chemo therapeutic. In another embodiment, the bacterial cells disclosed herein are administered to a subject twice daily. In another embodiment, the bacterial cells disclosed herein are administered to a subject in combination with a meal. In another embodiment, the bacterial cells disclosed herein are administered to a subject prior to a meal. In another embodiment, the bacterial cells disclosed herein are administered to a subject after a meal. The dosage of the pharmaceutical

composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

Treatment in vivo

[00607] The genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition caused by a toxic molecule, metabolite, or other deleterious molecule may be used (see, e.g., Takasuna et al., 1996; Nygard et al., 1994; Bowen et al., 2014). One exemplary animal model is a rodent model of chemotherapy drug-induced diarrhea. For example, the diarrhea is induced by administering irinotecan hydrochloride (CPT-11) in the drinking water and/or intravenously (Takasuna et al., 1996). Another exemplary animal model is the rat model of NS AID-induced diarrhea, for example, the diarrhea is induced by administering indomethacin orally (Nygard et al., 1994). The genetically engineered bacteria of the invention may be administered to the animal, e.g., by oral gavage, and treatment efficacy determined, e.g., by measuring

concentrations of the deleterious molecule and other byproducts in blood samples and in fecal samples.

[00608] In some embodiments, the genetically engineered bacteria of the invention is administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by endoscopy, colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. In some embodiments, the animal is sacrificed, and tissue samples are collected and analyzed, e.g., colonic sections are fixed and scored for inflammation and ulceration, and/or homogenized and analyzed for myeloperoxidase activity and cytokine levels (e.g. , IL- Ιβ, TNF-a, IL-6, IFN-γ and IL- 10).

Anti- inflammation and/or gut barrier function enhancer molecules

[00609] In addition to the gene sequence(s) and/or gene cassette(s) encoding one or more enzymes capable of detoxifying a deleterious molecule, the genetically engineered bacteria may further comprise one or more gene sequence(s) and/or gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene sequence(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene sequences are multiple copies of the same gene. In some embodiments, the two or more gene sequences are sequences encoding different genes. In some embodiments, the two or more gene sequences are sequences encoding multiple copies of one or more different genes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene cassettes are multiple copies of the same gene cassette. In some embodiments, the two or more gene cassettes are different gene cassettes for producing either the same or different anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the two or more gene cassettes are gene cassettes for producing multiple copies of one or more different anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the anti- inflammation and/or gut barrier function enhancer molecule is selected from the group consisting of a short-chain fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP- 1, IL- 10 (human or viral), IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, kynurenine, typtophan metabolite, indole, indole metabolite, a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-γ, IL- Ιβ, IL-6, IL-8, IL- 17, and/or chemokines, e.g., CXCL-8 and CCL2, AHR agonist (e.g. , indole acetic acid, indole-3-aldehyde, and indole), PXR agonist (e.g. , IP A), HDAC inhibitor (e.g. , butyrate), GPR41 and/or GPR43 activator (e.g. , butyrate and/or propionate and/or acetate), GPR109A activator (e.g., butyrate), inhibitor of NF- kappaB signaling (e.g., butyrate), modulator of PPARgamma (e.g. , butyrate), activator of AMPK signaling (e.g. , acetate), modulator of GLP- 1 secretion, and hydro xyl radical scavengers and antioxidants (e.g. , IP A). A molecule may be primarily anti- inflammatory, e.g. , IL- 10, or primarily gut barrier function enhancing, e.g. , GLP-2. Alternatively, a molecule may be both anti- inflammatory and gut barrier function enhancing.

[00610] In some embodiments, the genetically engineered bacteria of the invention express one or more anti- inflammation and/or gut barrier function enhancer molecule(s) that is encoded by a single gene, e.g. , the molecule is elafin and encoded by the PI3 gene, or the molecule is inter leukin- 10 and encoded by the IL10 gene. In alternate embodiments, the genetically engineered bacteria of the invention encode one or more an anti- inflammation and/or gut barrier function enhancer molecule(s), e.g. , butyrate, that is synthesized by a bio synthetic pathway requiring multiple genes.

[00611] The one or more gene sequence(s) and/or gene cassette(s) may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some

embodiments, expression from the plasmid may be useful for increasing expression of the anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the anti- inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the gene sequence(s)or gene cassette(s) for producing the anti- inflammation and/or gut barrier function enhancer molecule(s) is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the butyrate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle:

malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g. , Fig. 24 for exemplary insertion sites). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. Short chain Fatty Acids and Tryptophan Metabolites

[00612] One strategy in the treatment, prevention, and/or management of disorders may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti- inflammatory effectors.

[00613] For example, leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate, and those derived from tryptophan metabolism. These metabolites have been shown to play a major role in the prevention of inflammatory disease. As such one approach in the treatment, prevention, and/or management of gut barrier health may be to provide a treatment which contains one or more of such metabolites.

[00614] For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity (e.g. , as reviewed in

Thorburn et al., Diet, Metabolites, and "Western- Lifestyle" Inflammatory Diseases; Immunity Volume 40, Issue 6, 19 June 2014, Pages 833-842). (A) SCFA-induced promotion of mucus by gut epithelial cells, possibly through signaling through metabolite sensing GPCRs; (B) SCFA- induced secretion of IgA by B cells; (C) SCFA-induced promotion of tissue repair and wound healing; (D) SCFA-induced promotion of Treg cell development in the gut in a process that presumably facilitates immunological tolerance; (E) SCFA- mediated enhancement of epithelial integrity in a process dependent on inflammasome activation (e.g. , via NALP3) and IL- 18 production; and (F) anti- inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, 11-6, and IFN-gamma), and inhibition of NF-κΒ. Many of these actions of SCFAs in gut homeostatis can be ascribed to GPR43 and GPR109A, which are expressed by the colonic epithelium, by inflammatory leukocytes (e.g. neutrophils and marcophages) and by Treg cells. These receptors signal through G proteins, coupled to MAPK, PI3K and mTOR, as well as a separate arrestin- pathway, leading to NFkappa B inhibition. Other effects can be ascribed to SCFA-mediated HDAC inhibition, e.g. butyrate, which may regulate macrophage function and promote TReg cells.

[00615] In addition, a number of trptophan metabolites, including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehhyde, indole-3 propionic acid, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown to be essential for gut homeostais and promote gut-barrier health. These metabolites bind to aryl hydrocarbon receptor (Ahr). After agonist binding, AhR translocates to the nucleus, where it forms a heterodimer with AhR nuclear translocator (ARNT). AhR-dependent gene expression includes genes involved in the production of mediators important for gut homeostasis; these mediators include IL-22, antimicrobicidal factors, increased Thl7 cell activity, and the maintenance of intraepithelial lymphocytes and RORyt+ innate lymphoid cells.

[00616] Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3- dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis.

[00617] In addition, some indole metabolites, e.g., indole 3-propionic acid (IP A), may exert their effect an acitvating ligand of Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function, through downregulation of TLR4 signaling (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate

Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). As a result, indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health.

[00618] Thus, in some embodiments, the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryprophan metabolites.

Butyrate

[00619] In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes {see, e.g., Table 36 and Table 14). Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some

embodiments, the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from Peptoclostridium difficile, e.g., Peptoclostridium difficile strain 630: bcd.2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. In some embodiments, the genetically engineered bacteria comprise a

combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate. For example, in some embodiments, the genetically engineered bacteria comprise bcd.2, etfB3, etfA3, and thiAl from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.

Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB {e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.n some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

[00620] In some embodiments, additional genes may be mutated or knocked out, to further increase the levels of butyrate production. Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

[00621] Table 36 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.

[00622] Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 14. Table 14. Exemplary Polypeptide Sequences for Butyrate Production

[00623] The gene products of the bcd2, etfA3, and etfB3 genes in Clostridium difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant. In some embodiments, because the genetically engineered bacteria of the invention are designed to produce butyrate in a microaerobic or oxygen- limited environment, e.g., the mammalian gut, oxygen dependence could have a negative effect on butyrate production in the gut. It has been shown that a single gene from Treponema denticola {ter, encoding trans-2-enoynl-CoA reductase) can functionally replace this three-gene complex in an oxygen-independent manner. In some embodiments, the genetically engineered bacteria comprise a ter gene, e.g., from Treponema denticola, which can functionally replace all three of the bcd.2, etfB3, and etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the genetically engineered bacteria comprise thiAl, hbd, crt2, pbt, and buk, e.g., from

Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose..

[00624] In some embodiments, the genetically engineered bacteria of the invention comprise thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile; ter, e.g., from Treponema denticola; one or more of bcd.2, etfB3, and etfA3, e.g., from

Peptoclostridium difficile; and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00625] The gene products of pbt and buk convert butyrylCoA to Butyrate. In some embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA. In one embodiment, the genetically engineered bacteria comprise bcd.2, etfB3, etfA3, thiAl, hbd, and crt2, e.g., from Peptoclostridium difficile, and tesB from E. Coli and produce butyrate in low-oxygen conditions, in the presence of molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In one embodiment, the genetically engineered bacteria comprise ter gene (encoding iran5-2-enoynl-CoA reductase) e.g., from Treponema denticola, thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and tesB from E. Coli , and produce butyrate in low-oxygen conditions, in the presence of specific molecules or

metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00626] In some embodiments, the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis. In some embodiments, local butyrate production reduces gut inflammation, a symptom of IBD and other gut related disorders.

[00627] In one embodiment, the bcd.2 gene has at least about 80% identity with

SEQ ID NO: 141. In another embodiment, the bcd.2 gene has at least about 85% identity with SEQ ID NO: 141. In one embodiment, the bcd.2 gene has at least about 90% identity with SEQ ID NO: 141. In one embodiment, the bcd.2 gene has at least about 95% identity with SEQ ID NO: 141. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 141. Accordingly, in one embodiment, the bcd.2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 141. In another embodiment, the bcd.2 gene comprises the sequence of SEQ ID NO: 141. In yet another embodiment the bcd.2 gene consists of the sequence of SEQ ID NO: 141.

[00628] In one embodiment, the etfB3 gene has at least about 80% identity with

SEQ ID NO: 142. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 142. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 142. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 142. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 142. Accordingly, in one embodiment, the etfB3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 142. In another embodiment, the etfB3 gene comprises the sequence of SEQ ID NO: 142. In yet another embodiment the etfB3 gene consists of the sequence of SEQ ID NO: 142.

[00629] In one embodiment, the etfA3 gene has at least about 80% identity with

SEQ ID NO: 143. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 143. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 143. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 143. In another embodiment, the elf A3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 143. Accordingly, in one embodiment, the etfA3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 143. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 143. In yet another embodiment the etfA3 gene consists of the sequence of SEQ ID NO: 143.

[00630] In one embodiment, the thiAl gene has at least about 80% identity with

SEQ ID NO: 144. In another embodiment, the thiAl gene has at least about 85% identity with SEQ ID NO: 144. In one embodiment, the thiAl gene has at least about 90% identity with SEQ ID NO: 144. In one embodiment, the thiAl gene has at least about 95% identity with SEQ ID NO: 144. In another embodiment, the thiAl gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 144. Accordingly, in one embodiment, the thiAl gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 144. In another embodiment, the thiAl gene comprises the sequence of SEQ ID NO: 144. In yet another embodiment the thiAl gene consists of the sequence of SEQ ID NO: 144.

[00631] In one embodiment, the hbd gene has at least about 80% identity with

SEQ ID NO: 145. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 145. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 145. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 145. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 145. Accordingly, in one embodiment, the hbd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 145. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 145. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 145.

[00632] In one embodiment, the crt2 gene has at least about 80% identity with

SEQ ID NO: 146. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 146. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 146. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 146. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 146. Accordingly, in one embodiment, the crt2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 146. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 146. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 146.

[00633] In one embodiment, the pbt gene has at least about 80% identity with

SEQ ID NO: 147. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 147. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 147. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 147. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 147. Accordingly, in one embodiment, the pbt gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 147. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 147. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 147.

[00634] In one embodiment, the buk gene has at least about 80% identity with

SEQ ID NO: 148. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 148. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 148. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 148. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 148. Accordingly, in one embodiment, the buk gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 148. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 148. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 148. [00635] In one embodiment, the ter gene has at least about 80% identity with

SEQ ID NO: 149. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 149. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 149. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 149. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 149. Accordingly, in one embodiment, the ter gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 149. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 149. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 149.

[00636] In one embodiment, the tesB gene has at least about 80% identity with

SEQ ID NO: 150. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 150. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 150. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 150. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 150. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 150. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 150. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 150.

[00637] In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 35 through SEQ ID NO: 44. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 35 through SEQ ID NO: 44.

[00638] In some embodiments, one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In some

embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Peptoclostridicum difficile butyrate biosynthesis gene. The butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.

[00639] In some embodiments, the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production. In some embodiments, the local production of butyrate reduces food intake and ameliorates improves gut barrier function and reduces inflammation. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00640] In one embodiment, the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the butyrate gene cassette in nature.

[00641] In some embodiments, the butyrate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the butyrate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut.

Inducible promoters are described in more detail infra.

[00642] The butyrate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the butyrate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the butyrate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located on a plasmid in the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.

[00643] In some embodiments, the butyrate gene cassette is expressed on a low- copy plasmid. In some embodiments, the butyrate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of butyrate.

Propionate

[00644] In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing an ant i- inflammatory or gut barrier enhancer molecule, e.g., propionate, that is synthesized by a bio synthetic pathway requiring multiple genes and/or enzymes.

[00645] In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions. The genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 15, Table 16, Table 17, Table 18). Unmodified bacteria that are capable of producing propionate via an

endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. In some embodiments, the genetically engineered bacteria of the invention comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the genes pet, led, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC. In some

embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the Acul from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette comprises pet, IcdA, IcdB, IcdC, and acul. In another embodiment, the homolog of Acul in E coli, yhdH is used. This the propionate cassette comprises pet, IcdA, IcdB, IcdC, and yhdH. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA^, thrB, thrC, ilvA^, aceE, aceF, and Ipd, and optionally further comprise tesB. In another embodiment, the propionate gene cassette comprises the genes of the Sleepting Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm converts succinyl CoA to L- methylmalonylCoA, ygfG converts L-methylmalonylCoA into PropionylCoA, and ygfH converts propionylCoA into propionate and succinate into succinylCoA.

[00646] This pathway is very similar to the oxidative propionate pathway of

Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R- methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S- methylmalonyl-CoA via methymalonyl-CoA epimerase (GI: 18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, beep) which converts methylmalonyl-CoA to propionyl-CoA.

[00647] The genes may be codon-optimized, and translational and transcriptional elements may be added. Tables 15-17 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 18 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes. Table 15. Propionate Cassette Sequences (Acrylate Pathway)

Table 16. Propionate Cassette Sequences Sleeping Beauty Operon

[00648] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 15 (SEQ ID NO: 45 - SEQ ID NO: 59, and SEQ ID NO: 150) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 15 (SEQ ID NO: 45 - SEQ ID NO: 59, and SEQ ID NO: 150) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 15 (SEQ ID NO: 45 - SEQ ID NO: 59, and SEQ ID NO: 150) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 15 (SEQ ID NO: 45 - SEQ ID NO: 59, and SEQ ID NO: 150) or a functional fragment thereof.

[00649] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 16 (SEQ ID NO: 60 - SEQ ID NO: 63) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 16 (SEQ ID NO: 60 - SEQ ID NO: 63) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 16 (SEQ ID NO: 60 - SEQ ID NO: 63) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 16 (SEQ ID NO: 60 - SEQ ID NO: 63) or a functional fragment thereof.

[00650] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 17 (SEQ ID NO: 64 - SEQ ID NO: 69) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 17 (SEQ ID NO: 64 - SEQ ID NO: 69) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 17 (SEQ ID NO: 64 - SEQ ID NO: 69) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 17 (SEQ ID NO: 64 - SEQ ID NO: 69) or a functional fragment thereof.

[00651] Table 18 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria.

Table 18. Polypeptide Sequences for Propionate Synthesis

[00652] In some embodiments, the genetically engineered bacteria encode one or more polypeptide sequences of Table 18 (SEQ ID NO: 70-SEQ ID NO: 94, and SEQ ID

NO: 44) or a functional fragment or variant thereof. In some embodiments, genetically engineered bacteria comprise a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence of one or more polypeptide sequence of Table 18 (SEQ ID NO: 70-SEQ ID NO: 94, and SEQ ID NO: 44) or a functional fragment thereof.

[00653] In one embodiment, the bacterial cell comprises a non-native or heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a non-native or heterologous propionate gene cassette operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a propionate gene cassette, as well as at least one copy of a propionate gene cassette from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette.

[00654] Multiple distinct propionate gene cassettes are known in the art. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the propionate gene cassette is derived from an organism of the genus or species that includes, but is not limited to, Clostridium propionicum, Megasphaera elsdenii, or Prevotella ruminicola.

[00655] In one embodiment, the propionate gene cassette has been codon- optimized for use in the engineered bacterial cell. In one embodiment, the propionate gene cassette has been codon-optimized for use in Escherichia coli. In another embodiment, the propionate gene cassette has been codon-optimized for use in Lactococcus. When the propionate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more propionate than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat a disorder or condition caused by a toxic molecule, metabolite, or other deleterious molecule.

[00656] The present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme. As used herein, the term "functional fragment thereof or "functional variant thereof relates to an element having qualitative biological activity in common with the wild-type enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated propionate biosynthesis enzyme is one which retains essentially the same ability to synthesize propionate as the propionate biosynthesis enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having propionate biosynthesis enzyme activity may be truncated at the N- terminus or C-terminus, and the retention of propionate biosynthesis enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant. In another embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment.

[00657] As used herein, the term "percent (%) sequence identity" or "percent (%) identity," also including "homology," is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[00658] The present disclosure encompasses propionate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid

substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity,

hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, He, Asn, Gin, Phe, Cys, Pro, Trp, Tyr, Val).

[00659] In some embodiments, a propionate biosynthesis enzyme is

mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the propionate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

[00660] In one embodiment, the propionate biosynthesis gene cassette is from

Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium propionieum. In another embodiment, the propionate biosynthesis gene cassette is from a Megasphaera spp. In one embodiment, the Megasphaera spp. is Megasphaera elsdenii. In another embodiment, the propionate biosynthesis gene cassette is from Prevotella spp. In one embodiment, the

Prevotella spp. is Prevotella ruminicola. Other propionate biosynthesis gene cassettes are well-known to one of ordinary skill in the art.

[00661] In some embodiments, the genetically engineered bacteria comprise the genes pet, led, and aer from Clostridium propionieum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA^ , thrB, thrC, ilvA^ , aceE, aceF, and Ipd, and optionally further comprise tesB. The genes may be codon- optimized, and translational and transcriptional elements may be added. [00662] In one embodiment, the pet gene has at least about 80% identity with

SEQ ID NO: 45. In another embodiment, the pet gene has at least about 85% identity with SEQ ID NO: 45. In one embodiment, the pet gene has at least about 90% identity with SEQ ID NO: 45. In one embodiment, the pet gene has at least about 95% identity with SEQ ID NO: 45. In another embodiment, the pet gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. Accordingly, in one embodiment, the pet gene has at least about 80%, 821%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 921%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. In another embodiment, the pet gene comprises the sequence of SEQ ID NO: 45. In yet another embodiment the pet gene consists of the sequence of SEQ ID NO: 45.

[00663] In one embodiment, the IcdA gene has at least about 80% identity with

SEQ ID NO: 46. In another embodiment, the IcdA gene has at least about 85% identity with SEQ ID NO: 46. In one embodiment, the IcdA gene has at least about 90% identity with SEQ ID NO: 46. In one embodiment, the IcdA gene has at least about 95% identity with SEQ ID NO: 46. In another embodiment, the IcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46. Accordingly, in one embodiment, the IcdA gene has at least about 80%, 81%, 822%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 922%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46. In another embodiment, the IcdA gene comprises the sequence of SEQ ID NO: 46. In yet another embodiment the IcdA gene consists of the sequence of SEQ ID NO: 46.

[00664] In one embodiment, the IcdB gene has at least about 80% identity with

SEQ ID NO: 47. In another embodiment, the IcdB gene has at least about 85% identity with SEQ ID NO: 47. In one embodiment, the IcdB gene has at least about 90% identity with SEQ ID NO: 47. In one embodiment, the IcdB gene has at least about 95% identity with SEQ ID NO: 47. In another embodiment, the IcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47. Accordingly, in one embodiment, the IcdB gene has at least about 80%, 81%, 82%, 823%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 923%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47. In another embodiment, the IcdB gene comprises the sequence of SEQ ID NO: 47. In yet another embodiment the IcdB gene consists of the sequence of SEQ ID NO: 47.

[00665] In one embodiment, the IcdC gene has at least about 80% identity with

SEQ ID NO: 48. In another embodiment, the IcdC gene has at least about 85% identity with SEQ ID NO: 48. In one embodiment, the IcdC gene has at least about 90% identity with SEQ ID NO: 48. In one embodiment, the IcdC gene has at least about 95% identity with SEQ ID NO: 48. In another embodiment, the IcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. Accordingly, in one embodiment, the IcdA gene has at least about 80%, 81%, 82%, 83%, 824%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 924%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. In another embodiment, the IcdC gene comprises the sequence of SEQ ID NO: 48. In yet another embodiment the IcdC gene consists of the sequence of SEQ ID NO: 48.

[00666] In one embodiment, the etfA gene has at least about 80% identity with

SEQ ID NO: 49. In another embodiment, the etfA gene has at least about 825% identity with SEQ ID NO: 49. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 49. In one embodiment, the etfA gene has at least about 95% identity with SEQ ID NO: 49. In another embodiment, the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. Accordingly, in one embodiment, the etfA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. In another embodiment, the etfA gene comprises the sequence of SEQ ID NO: 49. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 49.

[00667] In one embodiment, the acrB gene has at least about 80% identity with

SEQ ID NO: 50. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 50. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 50. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO: 50. In another embodiment, the acrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 50. Accordingly, in one embodiment, the acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 50. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO: 50. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 50.

[00668] In one embodiment, the acrC gene has at least about 80% identity with

SEQ ID NO: 51. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 51. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 51. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO: 51. In another embodiment, the acrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 51. Accordingly, in one embodiment, the acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 51. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO: 51. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO:51.

[00669] In one embodiment, the thrA^ gene has at least about 280% identity with SEQ ID NO: 52. In another embodiment, the thrA^ gene has at least about 285% identity with SEQ ID NO: 52. In one embodiment, the thrA^ gene has at least about 90% identity with SEQ ID NO: 52. In one embodiment, the thrA^ gene has at least about 95% identity with SEQ ID NO: 52. In another embodiment, the thrA^ gene has at least about 96%, 97%, 928%, or 99% identity with SEQ ID NO: 52. Accordingly, in one embodiment, the thrA^ gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 82%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 928%, or 99% identity with SEQ ID NO: 52. In another embodiment, the thrA^ gene comprises the sequence of SEQ ID NO: 52. In yet another embodiment the thrA^ gene consists of the sequence of SEQ ID NO: 52.

[00670] In one embodiment, the thrB gene has at least about 80% identity with

SEQ ID NO: 53. In another embodiment, the thrB gene has at least about 85% identity with SEQ ID NO: 53. In one embodiment, the thrB gene has at least about 90% identity with SEQ ID NO: 53. In one embodiment, the thrB gene has at least about 95% identity with SEQ ID NO: 53. In another embodiment, the thrB gene has at least about 96%, 97%, 98%, or 92% identity with SEQ ID NO: 53. Accordingly, in one embodiment, the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. In another embodiment, the thrB gene comprises the sequence of SEQ ID NO: 53. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 53.

[00671] In one embodiment, the thrC gene has at least about 80% identity with

SEQ ID NO: 54. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 54. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 54. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 54. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. Accordingly, in one embodiment, the thrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO: 54. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO: 54.

[00672] In one embodiment, the ilvA^ gene has at least about 80% identity with

SEQ ID NO: 55. In another embodiment, the ilvA^ gene has at least about 85% identity with SEQ ID NO: 55. In one embodiment, the ilvA^ gene has at least about 90% identity with SEQ ID NO: 55. In one embodiment, the ilvA^ gene has at least about 95% identity with SEQ ID NO: 55. In another embodiment, the ilvA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. Accordingly, in one embodiment, the ilvA^ gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. In another embodiment, the ilvA^ gene comprises the sequence of SEQ ID NO: 55. In yet another embodiment the ilvA^ gene consists of the sequence of SEQ ID NO: 55.

[00673] In one embodiment, the aceE gene has at least about 80% identity with

SEQ ID NO: 56. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 56. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 56. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 56. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:56. Accordingly, in one embodiment, the aceE gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO: 56. In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO: 56.

[00674] In one embodiment, the aceF gene has at least about 80% identity with

SEQ ID NO: 57. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 57. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 57. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 57. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. Accordingly, in one embodiment, the aceF gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO: 57. In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO: 57.

[00675] In one embodiment, the Ipd gene has at least about 80% identity with

SEQ ID NO: 58. In another embodiment, the Ipd gene has at least about 85% identity with SEQ ID NO: 58. In one embodiment, the Ipd gene has at least about 90% identity with SEQ ID NO: 58. In one embodiment, the Ipd gene has at least about 95% identity with SEQ ID NO: 58. In another embodiment, the Ipd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. Accordingly, in one embodiment, the Ipd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. In another embodiment, the Ipd gene comprises the sequence of SEQ ID NO: 58. In yet another embodiment the Ipd gene consists of the sequence of SEQ ID NO: 58.

[00676] In one embodiment, the tesB gene has at least about 80% identity with

SEQ ID NO: 150. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 150. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 150. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 150. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 150. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 150. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 150. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 150.

[00677] In one embodiment, the acul gene has at least about 80% identity with

SEQ ID NO: 59. In another embodiment, the acul gene has at least about 85% identity with SEQ ID NO: 59. In one embodiment, the acul gene has at least about 90% identity with SEQ ID NO: 59. In one embodiment, the acul gene has at least about 95% identity with SEQ ID NO: 59. In another embodiment, the acul gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. Accordingly, in one embodiment, the acul gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. In another embodiment, the acul gene comprises the sequence of SEQ ID NO: 59. In yet another embodiment the acul gene consists of the sequence of SEQ ID NO: 59.

[00678] In one embodiment, the sbm gene has at least about 80% identity with

SEQ ID NO: 60. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 60. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 60. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 60. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. Accordingly, in one embodiment, the sbm gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. In another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 60. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 60. [00679] In one embodiment, the ygfD gene has at least about 80% identity with

SEQ ID NO: 61. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 61. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 61. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 61. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. Accordingly, in one embodiment, the ygfD gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO: 61. In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO: 61.

[00680] In one embodiment, the ygfG gene has at least about 80% identity with

SEQ ID NO: 62. In another embodiment, the ygfG gene has at least about 85% identity with SEQ ID NO: 62. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 62. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 62. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 62. Accordingly, in one embodiment, the ygfG gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 62. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO: 62. In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO: 62.

[00681] In one embodiment, the ygfH gene has at least about 80% identity with

SEQ ID NO: 63. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 63. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 63. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 63. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 63. Accordingly, in one embodiment, the ygfH gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 63. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO: 63. In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO: 63.

[00682] In one embodiment, the mutA gene has at least about 80% identity with

SEQ ID NO: 64. In another embodiment, the mutA gene has at least about 85% identity with SEQ ID NO: 64. In one embodiment, the mutA gene has at least about 90% identity with SEQ ID NO: 64. In one embodiment, the mutA gene has at least about 95% identity with SEQ ID NO: 64. In another embodiment, the mutA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 64. Accordingly, in one embodiment, the mutA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 64. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO: 64. In yet another embodiment the mutA gene consists of the sequence of SEQ ID NO: 64.

[00683] In one embodiment, the mutB gene has at least about 80% identity with

SEQ ID NO: 65. In another embodiment, the mutB gene has at least about 85% identity with SEQ ID NO: 65. In one embodiment, the mutB gene has at least about 90% identity with SEQ ID NO: 65. In one embodiment, the mutB gene has at least about 95% identity with SEQ ID NO: 65. In another embodiment, the mutB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 65. Accordingly, in one embodiment, the mutB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 65. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO: 65. In yet another embodiment the mutB gene consists of the sequence of SEQ ID NO: 65.

[00684] In one embodiment, the methymalonyl-CoA epimerase (GI 18042134) gene has at least about 80% identity with SEQ ID NO: 66. In another embodiment, the methymalonyl-CoA epimerase (GI 18042134) gene has at least about 85% identity with SEQ ID NO: 66. In one embodiment, the methymalonyl-CoA epimerase (GI 18042134) gene has at least about 90% identity with SEQ ID NO: 66. In one embodiment, the methymalonyl- CoA epimerase (GI 18042134) gene has at least about 95% identity with SEQ ID NO: 66. In another embodiment, the methymalonyl-CoA epimerase (GI 18042134) gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 66.. Accordingly, in one embodiment, the methymalonyl-CoA epimerase (GI 18042134) gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 66. In another embodiment, the methymalonyl-CoA epimerase (GI 18042134) gene comprises the sequence of SEQ ID NO: 66. In yet another embodiment the methymalonyl-CoA epimerase (GI 18042134) gene consists of the sequence of SEQ ID NO: 66.

[00685] In one embodiment, the mmdA gene has at least about 80% identity with

SEQ ID NO: 67. In another embodiment, the mmdA gene has at least about 85% identity with SEQ ID NO: 67. In one embodiment, the mmdA gene has at least about 90% identity with SEQ ID NO: 67. In one embodiment, the mmdA gene has at least about 95% identity with SEQ ID NO: 67. In another embodiment, the mmdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 67. Accordingly, in one embodiment, the mmdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 67. In another embodiment, the mmdA gene comprises the sequence of SEQ ID NO: 67. In yet another embodiment the mmdA gene consists of the sequence of SEQ ID NO: 67.

[00686] In one embodiment, the PFREUD_188870 gene has at least about 80% identity with SEQ ID NO: 68. In another embodiment, the PFREUD_188870 gene has at least about 85% identity with SEQ ID NO: 68. In one embodiment, the PFREUD_188870 gene has at least about 90% identity with SEQ ID NO: 68. In one embodiment, the PFREUD_188870 gene has at least about 95% identity with SEQ ID NO: 68. In another embodiment, the PFREUD_188870 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 68. Accordingly, in one embodiment, the PFREUD_188870 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 68. In another embodiment, the PFREUD_188870 gene comprises the sequence of SEQ ID NO: 68. In yet another embodiment the

PFREUD_188870 gene consists of the sequence of SEQ ID NO: 68.

[00687] In one embodiment, the Beep gene has at least about 80% identity with

SEQ ID NO: 69. In another embodiment, the Beep gene has at least about 85% identity with SEQ ID NO: 69. In one embodiment, the Beep gene has at least about 90% identity with SEQ ID NO: 69. In one embodiment, the Beep gene has at least about 95% identity with SEQ ID NO: 69. In another embodiment, the Beep gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 69. Accordingly, in one embodiment, the Beep gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 69. In another embodiment, the Beep gene comprises the sequence of SEQ ID NO: 69. In yet another embodiment the Beep gene consists of the sequence of SEQ ID NO: 69.

[00688] In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 70 through SEQ ID NO: 94. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 70 through SEQ ID NO: 94. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 70 through SEQ ID NO: 94. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 70 through SEQ ID NO: 94. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 70 through SEQ ID NO: 94. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 70 through SEQ ID NO: 94. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 70 through SEQ ID NO: 94. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or or more of SEQ ID NO: 70 through SEQ ID NO: 94.

[00689] In some embodiments, one or more of the propionate biosynthesis genes is a synthetic propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is an E. coli propionate biosynthesis gene. In some

embodiments, one or more of the propionate biosynthesis genes is a C. glutamicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a R. sphaeroides propionate biosynthesis gene. The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate.

[00690] In some embodiments, the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate. In some embodiments, one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production. In some embodiments, the local production of propionate reduces food intake and improves gut barrier function and reduces inflammation In some embodiments, the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00691] In one embodiment, the propionate gene cassette is directly operably linked to a first promoter. In another embodiment, the propionate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the propionate gene cassette in nature.

[00692] In some embodiments, the propionate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the propionate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the propionate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut.

Inducible promoters are described in more detail infra.

[00693] The propionate gene cassette may be present on a plasmid or

chromosome in the bacterial cell. In one embodiment, the propionate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the propionate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located on a plasmid in the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.

[00694] In some embodiments, the propionate gene cassette is expressed on a low-copy plasmid. In some embodiments, the propionate gene cassette is expressed on a high- copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of propionate. Acetate

[00695] In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and are capable of producing acetate. The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. Unmodified bacteria comprising acetate biosynthesis genes are known in the art and are capable of consuming various substrates to produce acetate under aerobic and/or anaerobic conditions (see, e.g., Ragsdale, 2008), and these endogenous acetate biosynthesis pathways may be a source of genes for the genetically engineered bacteria of the invention. In some embodiments, the genetically engineered bacteria of the invention comprise acetate

biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced. In some embodiments, the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli. In some embodiments, the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and/or Thermoacetogenium. The genetically engineered bacteria may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, the genetically engineered bacteria comprise a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing acetate. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid.

Tryptophan and Tryptophan Metabolism

Kynurenine

[00696] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine. Kynurenine is a metabolite produced in the first, rate-limiting step of tryptophan catabolism. This step involves the conversion of tryptophan to kynurenine, and may be catalyzed by the ubiquitously-expressed enzyme indoleamine 2,3-dioxygenase (IDO- 1), or by tryptophan dioxygenase (TDO), an enzyme which is primarily localized to the liver (Alvarado et al, 2015). Biopsies from human patients with IBD show elevated levels of IDO-1 expression compared to biopsies from healthy individuals, particularly near sites of ulceration (Ferdinande et al., 2008; Wolf et al., 2004). IDO-1 enzyme expression is similarly upregulated in trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced mouse models of IBD; inhibition of IDO-1 significantly augments the inflammatory response caused by each inducer (Ciorba et al., 2010; Gurtner et al, 2003; Matteoli et al., 2010). Kynurenine has also been shown to directly induce apoptosis in neutrophils (El-Zaatari et al., 2014). Together, these observations suggest that IDO- 1 and kynurenine play a role in limiting inflammation. The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise a gene or gene cassette for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase ant i- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions.

[00697] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase.

Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract {i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene, genes, or gene cassettes for producing kynurenic acid. In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions.

Tryptophan, Tryptophan Metabolism, and Tryptophan Metabolites

Tryptophan and the Kynurenine Pathway

[00698] Tryptophan (TRP) is an essential amino acid that, after consumption, is either incorporated into proteins via new protein synthesis, or converted a number of biologically active metabolites with a number of differing roles in health and disease (Perez-De La Cruz et al., 2007 Kynurenine Pathway and Disease: An Overview; CNS&Neurological Disorders -Drug Targets 2007, 6,398-410). Along one arm of tryptophan catabolism, trytophan is converted to the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) by tryptophan hydroxylase. Serotonin can further be converted into the hormone melatonin. A large share of tryptophan, however, is metabolized to a number of bioactive metabolites, collectively called kynurenines, along a second arm called the kynurenine pathway (KP). In the first step of catabolism, TRP is converted to Kynurenine, (KYN), which has well-documented immune suppressive functions in several types of immune cells, and has recently been shown to be an activating ligand for the arylcarbon receptor (AhR; also known as dioxin receptor). KYN was initially shown in the cancer setting as an endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival. In the gut, kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism.

[00699] More recently, additional tryptophan metabolites, collectively termed

"indoles", herein, including for example, indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc. which are generated by the microbiota, some by the human host, some from the diet, which are also able to function as AhR agonists, see e.g., Table 19 and FIG. 47 and elsewhere herein, and Lama et al, Nat Med. 2016 Jun;22(6):598-605; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands.

[00700] Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in additiona to kynurenine, tryptophan metabolites L-kynurenine, 6-formylindolcarbazole (FICZ, a photoproduct of TRP), and KYNA are have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-κΒ subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR- ARNT associated transcription of IL-6, which induced autocrine activation of IDOl via STAT3. This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer, supporting the idea that IDO/kynurenine-mediated

immunosuppression enables the immune escape of tumor cells.

[00701] In the gut, tryptophan may also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell respononse and promotion of Treg cells.

[00702] The rate-limiting conversion of TRP to KYN may be mediated by either of two forms of indoleamine 2, 3-dioxygenase (IDO) or by tryptophan 2,3-dioxygenase (TDO). One characteristic of TRP metabolism is that the rate-limiting step of the catalysis from TRP to KYN is generated by both the hepatic enzyme tryptophan 2,3-dioxygenase (TDO) and the ubiquitous expressed enzyme IDOl . TDO is essential for homeostasis of TRP concentrations in organisms and has a lower affinity to TRP than IDOl . Its expression is activated mainly by increased plasma TRP concentrations but can also be activated by glucocorticoids and glucagon. The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut (Sci Transl Med. 2013 July 10; 5(193): 193ra91). In some embodiments, the genetically engineered bacteria comprise one or more heterologous bacterially derived genes from Enterobacteriaceae, e.g. whose gene products catalyze the conversion of

TRP:KYN. Along one pathway, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA) which can antagonize glutamate receptors and can also bind AHR and also GPCRs, e.g. , GPR35, glutamate receptors, N-methyl D-aspartate

(NMD A) -receptors, and others. Along a third pathway of the KP, KYN can be converted to anthranilic acid (AA) and further downstream quinolinic acid (QUIN), which is a glutamate receptor agonist and has a neurotoxic role.

[00703] Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of a number of diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning trypophan and tryptophan metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites, and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.

Other Indole Tryptophan Metabolites

[00704] In addition to kynurenine and KYNA, numerous compounds have been proposed as endogenous AHR ligands, many of which are generated through pathways involved in the metabolism of tryptophan and indole (Bittinger et al., 2003; Chung and Gadupudi, 2011) A large number of metabolites generated through the tryptophan indole pathway are generated by microbiota in the gut. For example, bacteria take up tryptophan, which can be converted to mono-substituted indole compounds, such as indole acetic acid (IAA) and tryptamine, and other compounds, which have been found to activate the AHR (Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to sense microbiota- derived indoles; Nature Scientific Reoports 5: 12689).

[00705] In the gastronintestinal tract, diet derived and bacterially AhR ligands promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs (Spits et ah, 2013, Zelante et al, Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22; Immunity 39, 372-385, August 22, 2013).

[00706] Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of 11-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.

[00707] Table 19 lists exemplary tryptophan metabolites which have been shown to bind to AhR and which can be produced by the genetically engineered bacteria of the disclosure. Table 19. Indole Tryptophan Metabolites

[00708] In addition, some indole metabolites may exert their effect through

Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function. PXR-deficient (Nrli2-/-) mice showed a distinctly "leaky"gut physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). In particular, indole 3-propionic acid (IP A), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo.

[00709] As a result of PXR agonism, indole levels e.g. , produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. Ie., low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinaly barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health.

[00710] Although microbial degradation of tryptophan to indole-3-propionate has been shown in a numver of microorganisms (see, e.g., Elsden et al., The end products of the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr l ;107(3):283-8), to date, the bacterial entire bio synthetic pathway from tryptophan to IPA is unknown. In Clostridium sporogenes, tryptophan is catabolized via indole-3-pyruvate, indole-3-lactate, and indole- 3 -aery late to indole-3-propionate (O'Neill and DeMoss, Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys. 1968 Sep 20;127(l):361-9). Two enzymes that have been purified from C. sporogenes are tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss, Indolelactate dehydrogenase from Clostridium sporogenes, Can J Microbiol. 1968 Apr;14(4):429-35). Lactococcus lactis, catabolizes tryptophan by an aminotransferase to indole-3-pyruvate. In Lactobacillus casei and Lactobacillus helveticus tryptophan is also catabolized to indole- 3 -lactate through successive transamination and dehydrogenation (see, e.g. , Tryptophan catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-2077, and references therein).

[00711] L-tryptophan transaminase (e.g. , EC 2.6.1.27, e.g., Clostridium sporogenes or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indol- 3yl)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g.,

Clostridium sporogenes orLactobacillus casei) converts (indol-3yl) pyruvate and NADH and H+ to indole- 3 lactate and NAD+.

[00712] In some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes selected from tryptophan transaminase (e.g. , from C. sporogenes) and/or indole-3-lactate dehydrogenase (e.g. , from C. sporogenes), and/or indole-3-pyruvate aminotransferase (e.g. , from Lactococcus lactis). In other embodiments, such enzymes encoded by the bacteria are from Lactobacillus casei and/or Lactobacillus helveticus. [00713] In other embodiments, IPA producing circuits comprise enzymes depicted and described in FIG. 44 and elsewhere herein.

[00714] In some embodiments, the bacteria comprise gene sequence for producing one or more tryptophan metabolites, e.g., "indoles". In some embodiments, the bacteria comprise gene sequence for producing and indole selected from indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ. In some embodiments, the bacteria comprise gene sequence for producing an indole that functions as an AhR agonist, see e.g., Table 19 and FIG. 37.

[00715] In some embodiments, the genetically engineered bacteria comprise a circuit for the generation of IPA. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole-3- acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (Rubrivivax benzoatilyticus) and indole- 3 -aery late reductase (Clostridum botulinum). In some embodiments the expression of the gene sequences is under the control of an inducible promoter. Exemplary inducible promoters which may control the expression of the IPA biosynthetic cassette include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

[00716] In some embodiments, the bacteria comprise any one or more of the circuits described and depicted in Figures 48, 49A-H, 50A-E, 51A, 51B, 52A-E.

Methoxyindole pathway, Serotonin and Melatonin

[00717] The methoxyindole pathway leads to formation of serotonin (5-HT) and melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine synthesized in a two- step enzymatic reaction: First, enzymes encoded by one of two tryptophan hydroxylase genes (Tphl or Tph2) catalyze the rate-limiting conversion of tryptophan to 5-hydroxytryptophan (5- HTP), thus allocating the bioactivity of serotonin into either the brain (Tph2) or the periphery (Tphl). Then, 5-HTP undergoes decarboxylation to serotonin. Intestinal serotonin (5- hydroxytryptamine, 5-HT) is released by enterochromaffin cells and neurons and is regulated via the serotonin re-uptake transporter (SERT). The SERT is located on epithelial cells and neurons in the intestine. In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the intestine, e.g., decrease serotonin levels. [00718] 5-HT also functions a substrate for melatonin biosynthesis. The rate- limiting step of melatonin biosynthesis is 5-HT-N-acetylation resulting in the formation of N- acetyl- serotonin (NAS) with subsequent Omethylation into 5-methoxy-N-acetyltryptamine (melatonin). The deficient production of 5-HT, NAS, and melatonin contribute to depressed mood, disturbances of sleep and circadian rhythms. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle.

[00719] In certain embodiments, the genetically engineered bacteria influence 5-

HT synthesis, release, and/or degradation. Gut microbiota are interconnected with serotonin signaling and care capable of increasing serotonin levels through host serotonin production (Jano et al, Cell. 2015 Apr 9;161(2):264-76. doi: 10.1016/j.cell.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis). In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to ameliorate symptoms of inflammation. In some embodimetns, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In a non limiting example, serotonin can be converted to melatonin by, e.g., , tryptophan hydroxylase (TPH), hydro xyl-O- methyltransferase (HIOMT), N-acetyltransferase (NAT), aromatic -amino acid decarboxylase (AAAD). In some embodiments, the genetically engineered influence serotonin levels produced by the host.

[00720] In bacteria, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimic acid pathway. In these cells, synthesis starts with d- erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments the genetically engineered bacteria comprise an endogenous or exogenous cassette for the production of melatonin. As anon-limiting example, one pathway or cassette is described in Bochkov, Denis V.; Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011). "Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources". Journal of Chemical Biology 5 (1): 5-17. doi: 10.1007/sl2154-011-0064-8.

Exemplary Tryptophan and Tryptophan Metabolite Circuits

Decreasing Exogenous Tryptophan

[00721] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan and/or the level of a tryptophan metabolite. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. Tryptophan transporters may be expressed or modified in the recombinant bacteria described herein in order to enhance tryptophan transport into the cell. Specifically, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a tryptophan transporter which may be used to import tryptophan into the bacteria.

[00722] The uptake of tryptophan into bacterial cells is mediated by proteins well known to those of skill in the art. For example, three different tryptophan transporters, distinguishable on the basis of their affinity for tryptophan have been identified in E. coli (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake in bacteria. High affinity permease, Mtr, is negatively regulated by the trp repressor and positively regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17 and Heatwole et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is negatively regulated by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93).

[00723] In one embodiment, the at least one gene encoding a tryptophan transporter is a gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the bacterial cell described herein has been genetically engineered to comprise at least one heterologous gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli mtr gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli aroP gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli tnaB gene.

[00724] In some embodiments, the tryptophan transporter is encoded by a tryptophan transporter gene derived from a bacterial genus or species, including but not limited to, Escherichia, Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or

Corynebacterium glutamicum. In some embodiments, the bacterial species is Escherichia coli. In some embodiments, the bacterial species is Escherichia coli strain Nissle.

[00725] Assays for testing the activity of a tryptophan transporter, a functional variant of a tryptophan transporter, or a functional fragment of transporter of tryptophan are well known to one of ordinary skill in the art. For example, import of tryptophan may be determined using the methods as described in Shang et al. (2013) J. Bacteriol. 195:5334-42, the entire contents of each of which are expressly incorporated by reference herein.

[00726] In one embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import two-fold more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six- fold, seven- fold, eight-fold, nine-fold, ten- fold, fifteen- fold, twenty- fold, thirty- fold, fourty-fold, or fifty- fold, more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.

[00727] In addition to the tryptophan uptake transporters, in some embodiments, the genetically engineered bacteria further comprise a circuit for the production of tryptophan metabolites, as described herein, e.g., for the production of kynurenine, kynurenine

metabolites, or indole tryptophan metabolites as shown in Table 19.

[00728] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprise one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3- dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3- dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and

Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

[00729] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan, e.g., in combination with the production of indole metabolites, through expression of gene(s) and gene cassette(s) described herein. Increasing Kynurenine

[00730] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine.

[00731] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3- dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise on or more gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n- formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

[00732] The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase antiinflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00733] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. The genetically engineered bacteria may comprise any suitable gene for producing kynurenic acid. In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00734] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families

Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

[00735] In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low- oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. In some

embodiments, [00736] In some embodiments, the genetically engineered bacteria prevent the accumulation of post-kynurenine KP metabolites, e.g., neurotoxic metabolites, or diabetogenic metabolites. In some embodiments, the genetically engineered bacteria encode Kynureninase from Pseudomonas fluorescens.

[00737] In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYN ratio, e.g. in the circulation. In some embodiments the TRP:KYN ratio is increased. In some embodiments, TRP:KYN ratio is decreased. In some embodiments, the genetically engineered bacteria the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the

KYNA:QUIN ratio.

[00738] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Increasing Tryptophan

[00739] In some embodiments, the genetically engineered microorganisms of the present disclosure, are capable of producing tryptophan. Exemplary circuits for the production of tryptophan are shown in FIG. 41, FIG. 52A and FIG. 52B.

[00740] In some embodiments, the genetically engineered bacteria that produce tryptophan comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon. In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of E. coli. (Yanofsky, RNA (2007), 13: 1141-1154). In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of B.

subtilis. (Yanofsky, RNA (2007), 13: 1141-1154). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. coli. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from fi. subtilis.

[00741] Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, chorismate. Thus, in some embodiments, the genetically engineered bacteria optionally comprise sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding one or more enzymes of the tryptophan bio synthetic pathway and one or more gene sequences encoding one or more enzymes of the chorismate biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes.

[00742] In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 21).

Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD+ to NADH. As part of Tryptophan biosynthesis, E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved.

[00743] In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 21).

[00744] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. [00745] In any of these embodiments the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 21).

[00746] The inner membrane protein YddG of Escherichia coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al, FEMS Microbial Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

[00747] In some embodiments, the genetically engineered bacteria comprise a mechanism for metabolizing or degrading kyurenine, which, in some embodiments also results in the increased production of tryptophan. In some embodiments, the genetically engineered bacteria comprise sequence encoding the enzyme kynureninase. Kynureninase is produced to metabolize Kynurenine to Anthranilic acid in the cell. Schwarcz et al., Nature Reviews Neuroscience, 13, 465-477; 2012; Chen & Guillemin, 2009; 2; 1-19; Intl. J. Tryptophan Res. Exemplary kynureninase sequences are provided herein below in Table 22. In some embodiments, the engineered microbe has a mechanism for importing (transporting)

Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.

[00748] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes of the tryptophan biosynthetic pathway and sequence encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon, for example that of E. coli. or B. subtilis, and sequence encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes, for example, from E. Coli and sequence encoding kyureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes, for example from B. subtilis and sequence encoding kyureninase. In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, for example, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. Thus, in some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes, and sequence encoding

kyureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes, and sequence encoding kyureninase.

[00749] In some embodiments, the genetically engineered bacteria may optionally have a deletion or mutation in the endogenous trpE, rendering trpE non-functional. Accordingly, in one embodiment, the genetically engineered bacteria may comprise one or more gene(s) or gene cassette(s) encoding trpD, trpC, trpA, and trpD and kynureninase . This deletion may prevent tryptophan production through the endogenous chorismate pathway, and may increase the production of tryptophan from kynurenine through kynureninase.

[00750] In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 21) .

[00751] In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 21).

[00752] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function.

[00753] In any of these embodiments the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 21).

[00754] In any of these embodiments, the genetically engineered bacterium may further comprise gene sequence for exporting or secreting tryptophan from the cell. Thus, in some embodiments, the engineered bacteria further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG, an aromatic amino acid exporter. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene. In any of these embodiments, the genetically engineered bacterium may further comprise gene sequence for importing or transporting kynurenine into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.

[00755] In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan and/or kynureninase, under the control of a promoter that is activated by low-oxygen conditions, by inflammatory conditions, such as any of the promoters activated by said conditions and described herein. In some embodiments, the genetically engineered bacteria expresses one or more genes for producing tryptophan and/or kynureninase, under the control of a cancer- specific promoter, a tissue-specific promoter, or a constitutive promoter, such as any of the promoters described herein. Table 20 lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure.

Table 20. Tryptophan Synthesis Cassette Sequences

[00756] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 20 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 20 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 20 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence ofTable 20 or a functional fragment thereof.

[00757] In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 95 through SEQ ID NO: 107. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 95 through SEQ ID NO: 107. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 95 through SEQ ID NO: 107. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 95 through SEQ ID NO: 107. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 95 through SEQ ID NO: 107.

Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 95 through SEQ ID NO: 107. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 95 through SEQ ID NO: 107. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 95 through SEQ ID NO: 107.

[00758] Table 21 depicts exemplary polypeptide sequences feedback resistant

AroG and TrpE. Table 21 also depicts an exemplary TnaA (tryptophanase from E. coli) sequence. IN some embodiments, the sequence is encoded in circuits for tryptophan catabolism to indole; in other embodimetns, the sequence is deleted from the E coli chromosome to increase levels of tryptophan.

Table 21. Feedback resistant AroG and TrpE and tryptophanase sequences

[00759] In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 108 through SEQ ID NO: 111. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 108 through SEQ ID NO: 111. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 108 through SEQ ID NO: 111. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 108 through SEQ ID NO: 111. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 108 through SEQ ID NO: 111.

Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 108 through SEQ ID NO: 111. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 108 through SEQ ID NO: 111. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 108 through SEQ ID NO: 111.

[00760] Table 22 lists exemplary genes encoding kynureninase which are encoded by the genetically engineered bacteria of the disclosure in certain embodiments.

Table 22. Kynureninase protein sequences

[00761] In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 113 through SEQ ID NO: 115. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 113 through SEQ ID NO: 115. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 113 through SEQ ID NO: 115. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 113 through SEQ ID NO: 115. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 113 through SEQ ID NO: 115.

Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 113 through SEQ ID NO: 115. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 113 through SEQ ID NO: 115. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 113 through SEQ ID NO: 115.

[00762] Table 23 lists exemplary codon-optimized kynureninase cassette sequences.

Table 23. Selected codon-optimized kynureninase cassette sequences

The ptet-promoter is in bold, designed Ribosome binding site is underlined, codon- optimized protein coding sequence is in plain text, and the terminator is in italics.

[00763] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 23 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 23 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 23 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 23 or a functional fragment thereof.

[00764] In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 116 through SEQ ID NO: 118. In one embodiment, one or more

polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 116 through SEQ ID NO: 118. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 116 through SEQ ID NO: 118. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 116 through SEQ ID NO: 118. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 116 through SEQ ID NO: 118. Accordingly, in one embodiment, one or more polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 116 through SEQ ID NO: 118. In another embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 116 through SEQ ID NO: 118. In another embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria consists of the sequence of one or more of SEQ ID NO: 116 through SEQ ID NO: 118. [00765] The genetically engineered bacteria may comprise any suitable gene for producing kynureninase. In some embodiments, the gene for producing kynureninase is modified and/or mutated, e.g., to enhance stability, increase kynureninase production. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00766] The genetically engineered bacteria may comprise any suitable gene for producing kynureninase. In some embodiments, the gene for producing kynureninase is modified and/or mutated, e.g., to enhance stability, increase kynureninase production. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase in low-oxygen conditions. In some

embodiments, the genetically engineered bacteria are capable of producing kynureninase in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

Producing Kynurenic Acid

[00767] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e. , the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene or genes for producing kynurenic acid. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (also referred to as kynurenine aminotransferases (e.g. , KAT I, II, III)).

[00768] In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g. , to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00769] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenic acid, which are bacterially derived. In some embodiments, the enzymes for producing kynureic acid are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

[00770] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters, gene sequence(s) encoding kynureninase, and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding one or more kynurenine aminotransferases.

[00771] In some embodiments, the one or more genes for producing kynurenic acid are modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g. , comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00772] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g. , high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g. , thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. Producing Indole Tryptophan Metabolites and Tryptamine

Tryptamine

[00773] In some embodiments the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is derived from the direct decarboxylation of tryptophan. Tryptophan is converted to indole- 3 -acetic acid (IAA) via the enzymes tryptophan monooxygenase (laaM) and indole-3- acetamide hydrolase (laaH), which constitute the indole- 3 -acetamide (IAM) pathway, see eg., FIG. 53, FIG. 47A and FIG. 47B.

[00774] A non- limiting example of such as strain is shown in FIG. 49. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s). e.g., from Catharanthus roseus. In one embodiment the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s). e.g., from Catharanthus roseus. In one embodiment the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from Ruminococcus Gnavus.

[00775] Another non-limiting example of such as strain is shown in FIG. 52C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus.

[00776] In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Indole-3 -acetaldehyde and FICZ

[00777] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole- 3 -acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole- 3 -acetaldehyde and FICZ from tryptophan are shown in FIG. 49B.

[00778] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In one

embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g. , from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and ipdC.

[00779] Further exemplary gene cassettes for the production of produce indole-3- acetaldehyde and FICZ from tryptophan are shown in FIG. 49C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA.

[00780] In any of these embodiments, the genetically engineered bacteria which produce produce indole- 3 -acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole- 3 -acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole- 3 -acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Indole-3-acetonitrile

[00781] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetonitrile from tryptophan. A non-limiting example of such gene sequence(s) which allow in which the genetically engineered bacteria to produce indole-3- acetonitrile from tryptophan is depicted in FIG. 49D.

[00782] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 (indoleacetaldoxime dehydratase).

[00783] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71al3. [00784] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3.

[00785] In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Kynurenine

[00786] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenine from tryptophan. Non- limiting example of such gene sequence(s) are shown FIG. 49E and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ID01(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDOl from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine— oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in

combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.

[00787] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.

[00788] In any of these embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Kynureninic acid

[00789] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynureninic acid from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 49F and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ID01(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDOl from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene

sequence(s) which encode cclbl and/or cclb2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine— oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cclb2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.

[00790] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha- aminoadipate aminotransferase, mitochondrial).

[00791] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB l (Kynurenine— oxoglutarate transaminase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB l from homo sapiens). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine— oxoglutarate transaminase 3) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cclbl and/or cclb2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of . cclbl and/or cclb2 and/or aadat and/or got2. [00792] In any of these embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene

sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Indole

[00793] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole from tryptophan. Non- limiting example of such gene sequence(s) are shown FIG. 49G and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA (tryptophanase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA from E. coli.

[00794] In any of these embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Other indole metabolites

[00795] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. Non-limiting example of such gene sequence(s) are shown FIG. 49G and described elsewhere herein. In one

embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 (myrosinase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 from Arabidopsis thaliana.

[00796] In any of these embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Indole acetic acid

[00797] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole- 3 -acetic acid.

[00798] Non-limiting example of such gene sequence(s) are shown in FIG. 50A,

FIG. 50B, FIG. 50C, FIG. 50D, and FIG. 50E.

[00799] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from S. cerevisae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate

aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L- tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole- 3 -acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl (Indole- 3 -acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3- pyruvate decarboxylase, e.g. , from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3- pyruvate decarboxylase, e.g. , from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or staO and/or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from iadl and/or aao l.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or staO and in combination with one or more sequences encoding enzymes selected from iadl and/or aao l (see, e.g., FIG. 50A).

[00800] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 50B. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole- 3 -acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl (Indole- 3 -acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iadl and/or aao l . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iadl and/or aao l. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iadl and/or aao l .

[00801] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 52D. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g. , from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and/or ipdC and/or iadl . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and ipdC and iadl .

[00802] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 50C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole-3-pyruvate

monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from Enterobacter cloacae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L- tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and yuc2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and yuc2.. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and yuc2.

[00803] Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 50D. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH

(Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH.

[00804] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 50E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nitl (Nitrilase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nitl from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH

(Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi).In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71al3. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nitl and/or iaaH. In one

embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3, and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3 and nitl and iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3 and nitl and iaaH.

[00805] In any of these embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some

embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

Indole -3 -propionic acid (IPA)

[00806] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-propionic acid from tryptophan. FIG. 51A and FIG 51B depict schematics fexemplary circuits for the production of indole-3-propionic acid.

[00807] In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase from Rubrivivax benzoatilyticus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole- 3 -aery late reductase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole- 3 -aery late reductase from Clostridum botulinum. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole-3-acrylate reductase.

[00808] FIG. 52E depicts another non-limiting example of an indole-3- propionate-producing strain. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA (indole-3-propionyl- CoA:indole-3-lactate CoA transferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC (indole-3-lactate dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD (indole-3-acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding Acul (acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding Acul from Rhodobacter sphaeroides. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldHl (3-lactate dehydrogenase 1). In some

embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldHl from Clostridium sporogenes,. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 (indole- 3 -lactate dehydrogenase 2). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 from Clostridium sporogenes). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acul and/or fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acul and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acul and fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acul and fldH2.

[00809] In any of these embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 41, FIG. 52A and/or FIG. 52B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole-3- propionic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[00810] In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 different tryptophan metabolites. In certain embodiments the bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites selected from tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3-propionic acid.

[00811] In in any of these embodiments the expression of the gene sequences for the production of the indole and other tryptophan metabolites, including, but not limited to, tryptamine and/or indole-3 acetaladehyde, indole-3 acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3-propionic acid is under the control of an inducible promoter. Exemplary inducible promoters which may control the expression of the bio synthetic cassettes include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

[00812] Exemplary circuits for the production of indole metabolites/derivatives are shown in FIG. 49A through FIG. 49H, FIG. 50A through FIG. 50E, and FIG. 51A though FIG 51B, and FIG. 52A through FIG. 52E.

Table 24. Non-limiting examples of Sequences for Tryptophan to tryptamine conversion

[00813] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 24 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 24 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 24 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 24 or a functional fragment thereof.

[00814] In one embodiment, the Tryptophan Decarboxylase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 119 or SEQ ID NO: 120: . In another embodiment, the Tryptophan Decarboxylase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 119 or SEQ ID NO: 120. In one embodiment, the Tryptophan Decarboxylase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 119 or SEQ ID NO: 120. In one embodiment, the Tryptophan Decarboxylase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 119 or SEQ ID NO: 120. In another embodiment, the Tryptophan Decarboxylase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 119 or SEQ ID NO: 120. Accordingly, in one embodiment, the Tryptophan Decarboxylase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 119 or SEQ ID NO: 120. In yet another embodiment the Tryptophan Decarboxylase gene consists of the sequence of SEQ ID NO: 119 or SEQ ID NO: 120.

[00815] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole Acetic Acid, e.g. , via a tryptophan aminotransferase cassette. A non- limiting example of such a tryptophan aminotransferase expressed by the genetically engineered bacteria is in Table 25. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter, and further produce Indole-3-aldehyde and Indole Acetic Acid from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan and/or indole metabolite exporter. [00816] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 25 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 25 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 25 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 25 or a functional fragment thereof.

[00817] In one embodiment, the Trp aminotransferase gene has at least about

80% identity with the entire sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In another embodiment, the Trp aminotransferase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In one embodiment, the Trp

aminotransferase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In one embodiment, the Trp aminotransferase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In another embodiment, the Trp aminotransferase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 121 or SEQ ID NO: 122. Accordingly, in one embodiment, the Trp aminotransferase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In another embodiment, the Trp aminotransferase gene comprises the sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In yet another embodiment the Trp aminotransferase gene consists of the sequence of SEQ ID NO: 121 or SEQ ID NO: 122.

[00818] The genetically engineered bacteria may comprise any suitable gene for producing Indole- 3 -aldehyde and/or Indole Acetic Acidand/or Tryptamine. In some

embodiments, the gene for producing kynurenine is modified and/or mutated, e.g. , to enhance stability, increase Indole- 3 -aldehyde and/or Indole Acetic Acidand/or Tryptamine production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite , e.g. , comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole- 3 -aldehyde and/or Indole Acetic Acidand/or Tryptamine under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[00819] Table 26 comprises polypeptide sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure.

Table 26. Tryptophan Pathway Catabolic Enzymes

[00820] In one embodiment, the tryptophan pathway catabolic enzyme has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 123 through SEQ ID NO: 150. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150. Accordingly, in one embodiment, the tryptophan pathway catabolic enzyme has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150. In another embodiment, the tryptophan pathway catabolic enzyme comprises the sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150. In yet another embodiment the tryptophan pathway catabolic enzyme consists of the sequence of one or more SEQ ID NO: 123 through SEQ ID NO: 150.

[00821] In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In som embodiments the bacteria further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptamine exporter. In some embodiments the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

Indole 3 -propionic acid (IPA)

[00822] In some embodiments, the genetically engineered bacteria comprise at least one genetic circuit for the producton of indole-3-propionate (IPA). In some embodiments, the indole-3-propionate-producing strain optionally produces tryptophan from a chorismate precursor, and the strain optionally comprises additional circuits for tryptophan production and/or tryptophan uptake/transport s described herein. Additionally the genetically engineered bacteria comprise a circuit, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indol-3yl)pyruvate from tryptophan), fldA (indole-3- propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or Acul: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole- 3-propionyl-CoA). The circuits further comprise fldHl and/or fldH2 (indole- 3 -lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3- yl)pyruvate into indole- 3 -lactate).

[00823] Table 27 depicts non-limiting examples of contemplated polypeptide sequences, which are encoded by the indole-3-propionate producing bacteria.

Table 27. Non-limiting Examples of Sequences for indole-3-propionate Production

[00824] In one embodiment, the tryptophan pathway catabolic enzyme has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 151 through SEQ ID NO: 157. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157. Accordingly, in one embodiment, the tryptophan pathway catabolic enzyme has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157. In another embodiment, the tryptophan pathway catabolic enzyme comprises the sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157. In yet another embodiment the tryptophan pathway catabolic enzyme consists of the sequence of one or more SEQ ID NO: 151 through SEQ ID NO: 157.

[00825] In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of one or more indole pathway metabolites described herein from tryptophan or a tryptophan metabolite. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the genetically engineered bacteria additionally produce tryptophan and/or chorismate through any of the pathways described herein, e.g. FIG. 41, FIG. 52A and FIG. 52B. In some embodiments the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

[00826] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose or tetracycline. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. In some embodiments, the tryptophan synthesis and/or tryptophan catabolism cassette(s) is under control of an inducible promoter. Exemplary inducible promoters which may control the expression of the al teast one sequence(s) include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

[00827] Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more exporters for exporting biological molecules or substrates, such any of the exporters described herein or otherwise known in the art, (6) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (7) combinations of one or more of such additional circuits.

Tryptophan Repressor (TrpR)

[00828] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, e.g., sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC.

Tryptophan and Tryptophan MetaboliteTransport

[00829] Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell.

[00830] The inner membrane protein YddG of E. coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al, FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

[00831] In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.

[00832] In some embodiments the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP.

[00833] In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transporte gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.

[00834] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[00835] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[00836] In some embodiments, the native transporter gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome.

[00837] In some embodiments, the genetically engineered bacterium is E. coli

Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[00838] In some embodiments, the genetically engineered bacterium is E. coli

Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g. , Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter.

Secreted Polypeptides

IL-10

[00839] In some embodiments, the genetically engineered bacteria of the invention are capable of producing IL- 10. Interleukin- 10 (IL- 10) is a class 2 cytokine, a category which includes cytokines, interferons, and interferon- like molecules, such as IL- 19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, IL-29, IFN-a, IFN-β, IFN-δ, IFN-ε, IFN-K, IFN-τ, IFN-co, and limitin. IL- 10 is an anti- inflammatory cytokine that signals through two receptors, IL- 10R1 and IL- 10R2. Anti- inflammatory properties of human IL- 10 include down-regulation of pro-inflammatory cytokines, inhibition of antigen presentation on dendritic cells or suppression of major histocompatibility complex expression. Deficiencies in IL- 10 and/or its receptors are associated with IBD and intestinal sensitivity (Nielsen, 2014). Bacteria expressing IL- 10 or protease inhibitors may ameliorate conditions such as Crohn' s disease and ulcerative colitis (Simpson et al., 2014). The genetically engineered bacteria may comprise any suitable gene encoding IL- 10, e.g. , human IL- 10. In some embodiments, the gene encoding IL- 10 is modified and/or mutated, e.g. , to enhance stability, increase IL- 10 production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing IL- 10 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL- 10 in low- oxygen conditions. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that encodes IL- 10. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence comprising SEQ ID NO: 158 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence comprising SEQ ID NO: 73 or a nucleic acid sequence comprising SEQ ID NO: 158 or a functional fragment thereof. Table 28. Nucleic Acid Sequence of IL-10 (SEQ ID NO: 158) monomerized human IL-10 (SEQ ID NO: 325), and viral IL-10 (SEQ ID NOs: 326 and 327)

[00840] Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the dimerization of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements (see, e.g., Josephson, K. et al. Design and analysis of an engineered human interleukin-10 monomer. J. Biol. Chem. 275, 13552-13557 (2000), and Yoon, S. I. et al. Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. J. Biol. Chem. 287, 26586-26595 (2012). Monomoerized IL-10 therefore comnprises a small linker which deviates from the wild-type human IL-10 sequence. This linker causes the IL10 to become active as a monomer rather than a dimer (see, e.g., Josephson, K. et al. Design and analysis of an engineered human inter leukin- 10 monomer. J. Biol. Chem. 275, 13552-13557 (2000), and Yoon, S. I. et al. Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. J. Biol. Chem. 287, 26586- 26595 (2012)).

[00841] Secretion of a monomeric protein may have advantages, avoiding the extra step of dimerization in the periplasmic space. Moreover, there is more flexibility in the selection of appropriate secretion systems. For example, the tat-dependent secretion system secretes polypeptides in a folded fashion. Dimers cannot fold correctly without the formation of disulfide bonds. Disulfide bonds, however, cannot form in the reducing intracellular environment and require the oxidizing environment of the periplasm to form. Therefore, the tat-dependent system may no be appropriate for the secretion of proteins which require dimerization to function properly.

[00842] In some embodiments, the genetically engineered bacteria of the invention are capable of producing monomerized human IL-10. In some embodiments, the genetically engineered bacteria are capable of producing monomerized IL-10 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing monomerized IL-10 in low-oxygen conditions. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that encodes monomerized IL-10. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence comprising SEQ ID NO: 325 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence comprising SEQ ID NO: 325 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a sequence which encodes the polypeptide encoded by SEQ ID NO: 325 or a fragment or functiona variant thereof. In some embodiments, the monomerized IL-10 expressed by the bacteria stimulates IL-10R1 and IL-10R2 and initiates signal transduction. Signaling includes Stat signaling, e.g. through the phosphorylation of Tyr705 and/or Ser727.

[00843] In some embodiments, the genetically engineered bacteria of the invention are capable of producing viral IL-10. Exemplary viral IL-10 homologues encoded by the bacteria include human cytomegalo- (HCMV) and Epstein-Barr virus (EBV) IL-10. Apart from its anti- inflammatory effects, human IL-10 also possesses pro-inflammatory activity, e.g., stimulation of B-cell maturation and proliferation of natural killer cells (Foerster et al, Secretory expression of biologically active human Herpes virus inter leukin- 10 analogues in Escherichia coli via a modified Sec-dependent transporter construct, BMC Biotechnol. 2013; 13: 82, and references therein). In contrast, viral IL-10 homologues share many biological activities of hIL-10 but, due to selective pressure during virus evolution and the need to escape the host immune system, also display unique traits, including increased stability and lack of immuno stimulatory functions (Foerster et al, and references therein). As such, viral counterparts may be useful and possibly more effective than hIL-10 with respect to antiinflammatory and/or immune suppressing effects.

[00844] In some embodiments, the genetically engineered bacteria are capable of producing viral IL-10 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing viral IL-10 in low-oxygen conditions. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that encodes viral IL-10. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence comprising SEQ ID NO: 326 and/or SEQ ID NO: 327 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence comprising SEQ ID NO: 326 and/or SEQ ID NO: 327 or a functional fragment thereof. In some embodiments, the viral d IL-10 expressed by the bacteria stimulates IL-10R1 and IL-10R2 and initiates signal transduction. Signaling includes Stat signaling, e.g. through the phosphorylation of Tyr705 and/or Ser727.

IL-2

[00845] In some embodiments, the genetically engineered bacteria are capable of producing IL-2. Interleukin 2 (IL-2) mediates autoimmunity by preserving health of regulatory T cells (Treg). Treg cells, including those expressing Foxp3, typically suppress effector T cells that are active against self- antigens, and in doing so, can dampen autoimmune activity. IL-2 functions as a cytokine to enhance Treg cell differentiation and activity while diminished IL-2 activity can promote autoimmunity events. IL-2 is generated by activated CD4+ T cells, and by other immune mediators including activated CD8+ T cells, activated dendritic cells, natural killer cells, and NK T cells. IL-2 binds to IL-2R, which is composed of three chains including CD25, CD122, and CD132. IL-2 promotes growth of Treg cells in the thymus, while preserving their function and activity in systemic circulation. Treg cell activity plays an intricate role in the IBD setting, with murine studies suggesting a protective role in disease pathogenesis. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 159 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 159 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing IL-2 under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-2 in low- oxygen conditions.

Table 29. SEQ ID NO: 159

IL-22

[00846] In some embodiments, the genetically engineered bacteria are capable of producing IL-22. Interleukin 22 (IL-22) cytokine can be produced by dendritic cells, lymphoid tissue inducer-like cells, natural killer cells and expressed on adaptive lymphocytes. Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of 11-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function. IL-22' s association with IBD susceptibility genes may modulate phenotypic expression of disease as well. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 160 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 160 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing IL-22 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-22 in low- oxygen conditions.

Table 30. SEQ ID NO: 160

IL-27

[00847] In some embodiments, the genetically engineered bacteria are capable of producing IL-27. Interleukin 27 (IL-27) cytokine is predominately expressed by activated antigen presenting cells, while IL-27 receptor is found on a range of cells including T cells, NK cells, among others. In particular, IL-27 suppresses development of pro-inflammatory T helper 17 (Thl7) cells, which play a critical role in IBD pathogenesis. Further, IL-27 can promote differentiation of IL- 10 producing Trl cells and enhance IL- 10 output, both of which have antiinflammatory effects. IL-27 has protective effects on epithelial barrier function via activation of MAPK and STAT signaling within intestinal epithelial cells. Additionally, IL-27 enhances production of antibacterial proteins that curb bacterial growth. Improvement in barrier function and reduction in bacterial growth suggest a favorable role for IL-27 in IBD pathogenesis. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 161 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 161 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing IL-27 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-27 in low- oxygen conditions. Table 31. SEQ ID NO: 161

SOD

[00848] In some embodiments, the genetically engineered bacteria of the invention are capable of producing SOD. Increased ROS levels may lead to enhanced expression of vascular cell adhesion molecule 1 (VCAM- 1), which can facilitate translocation of inflammatory mediators to disease affected tissue, and result in a greater degree of inflammatory burden. Antioxidant systems including superoxide dismutase (SOD) can function to mitigate overall ROS burden. However, studies indicate that the expression of SOD in the setting of IBD may be compromised, e.g. , produced at lower levels in IBD, thus allowing disease pathology to proceed. Further studies have shown that supplementation with SOD to rats within a colitis model is associated with reduced colonic lipid peroxidation and endothelial VCAM- 1 expression as well as overall improvement in inflammatory environment. Thus, in some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 162 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 162 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing SOD under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing SOD in low-oxygen conditions.

Table 32. SEQ ID NO: 162

GLP2

[00849] In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 or proglucagon. Glucagon- like peptide 2 (GLP-2) is produced by intestinal endocrine cells and stimulates intestinal growth and enhances gut barrier function. GLP-2 administration has therapeutic potential in treating IBD, short bowel syndrome, and small bowel enteritis (Yazbeck et al., 2009). The genetically engineered bacteria may comprise any suitable gene encoding GLP-2 or proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments, a protease inhibitor, e.g. , an inhibitor of dipeptidyl peptidase, is also

administered to decrease GLP-2 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et al., 2009). In some embodiments, the gene encoding GLP-2 or proglucagon is modified and/or mutated, e.g. , to enhance stability, increase GLP-2 production, and/or increase gut barrier enhancing potency under inducing conditions. In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-2 or proglucagon under inducing conditions. GLP-2 administration in a murine model of IBD is associated with reduced mucosal damage and inflammation, as well as a reduction in inflammatory mediators, such as TNF-a and IFN-y. Further, GLP-2 supplementation may also lead to reduced mucosal myeloperoxidase in colitis/ileitis models. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 163 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 163 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 in low-oxygen conditions.

Table 33. SEQ ID NO: 163 GLP-2

[00850] In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 analogs, including but not limited to, Gattex and teduglutide. Teduglutide is a protease resistan analog of GLP-2. It is made up of 33 amino acids and differs from GLP-2 by one amino acid (alanine is substituted by glycine). The significance of this substitution is that teduglutide is longer acting than endogenous GLP-2 as it is more resistant to proteolysis from dipeptidyl peptidase-4.

Table 34. SE ID NO: 164 Tedu lutide

[00851] In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 164 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 164 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing

Teduglutide in low-oxygen conditions.

IL-19, IL-20, and/or IL-24

[00852] In some embodiments, the genetically engineered bacteria are capable of producing IL-19, IL-20, and/or L-2A. In some embodiments, the genetically engineered bacteria are capable of producing IL-19, IL-20, and/or IL-24 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-19, IL-20 and/or IL-24 in low-oxygen conditions.

Inhibition of pro-inflammatory molecules

[00853] In some embodiments, the genetically engineered bacteria of the invention are capable of producing a molecule that is capable of inhibiting a pro-inflammatory molecule. The genetically engineered bacteria may express any suitable inhibitory molecule, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA, that is capable of neutralizing one or more pro-inflammatory molecules, e.g., TNF, IFN-γ, IL-Ιβ, IL-6, IL-8, IL-17, IL-18, IL-21, IL-23, IL-26, IL-32, Arachidonic acid, prostaglandins {e.g., PGE ₂ ), PGI ₂ , serotonin, thromboxanes (e.g. , TXA ₂ ), leukotrienes (e.g., LTB ₄ ), hepoxillin A3, or chemokines (Keates et al., 2008; Ahmad et al., 2012). The genetically engineered bacteria may inhibit one or more pro-inflammatory molecules, e.g. , TNF, IL- 17. In some embodiments, the genetically engineered bacteria are capable of modulating one or more molecule(s) shown in Table 35. In some embodiments, the genetically engineered bacteria are capable of inhibiting, removing, degrading, and/or metabolizing one or more inflammatory molecules.

Table 35

[00854] In some embodiments, the genetically engineered bacteria are capable of producing an anti- inflammation and/or gut barrier enhancer molecule and further producing a molecule that is capable of inhibiting an inflammatory molecule. In some embodiments, the genetically engineered bacteria of the invention are capable of producing an anti- inflammation and/or gut barrier enhancer molecule and further producing an enzyme that is capable of degrading an inflammatory molecule. For example, the genetically engineered bacteria of the invention are capable of expressing a gene cassette for producing butyrate, as well as a molecule or biosynthetic pathway for inhibiting, removing, degrading, and/or metabolizing an inflammatory molecule, e.g., PGE ₂ .

Combinations of Detox Effectors and Gut Barrier Enhancers

[00855] In some embodiments, the bacterial cell produces a first payload that is capable of detoxifying a deleterious molecule. In some embodiments, the bacterial cell produces a second payload that is capable of enhancing gut barrier function and anti- inflammation.

[00856] In some embodiments, the first payload is carboxypeptidase Gi (CPD

Gi) or carboxypeptidase G ₂ (CPD G ₂ ). In some embodiments, the payload is D-saccharic acid 1, 4-lactone (SAL). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38). In some embodiments, the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen.. In some embodiments, the payload is a proton pump inhibitor. In some embodiments, the payload is a heavy metal chelator. In some embodiments, the payload is a plant phytochelatin. In some embodiments, the payload is a short-chained fatty acid, e.g. butyrate, propionate, or acetate. In some embodiments, the payload is the enzyme Pseudomonas.

[00857] In some embodiments, the second payload is selected from butyrate, propionate, acetate, IL- 10, IL-2, IL-22, IL-27, IL-20, IL-24, IL- 19, SOD, GLP2, IFN-γ, TNF-a, 1L- 1B, or tryptophan and/or its metabolites.

[00858] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. ,

carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. ,

carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., Ih-Π. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. ,

carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00859] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ) in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00860] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D- saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4- lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00861] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10- hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g.,, IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00862] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00863] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in

combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in

combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00864] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor) in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, t a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00865] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00866] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant

phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00867] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00868] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00869] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00870] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., 1L-1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00871] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., Ih-Π. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., 1L-1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.

[00872] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00873] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G ₂ (CPD G ₂ ), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G ₂ (CPD G ₂ ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00874] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D-saccharic acid 1, 4-lactone (SAL), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co -administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4- lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00875] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00876] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00877] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g.,, IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00878] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00879] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00880] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00881] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

[00882] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some

embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00883] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.

[00884] In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is coacetatered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. propionate, and is coacetatered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN-γ. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.

Nucleic Acids

[00885] In some embodiments the genetically engineered bacteria comprises a nucleic acid encoding one of an effector molecule or biosynthetic cassette for producing a payload that is capable of detoxifying a deleterious molecule. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthesis cassette for producing a payload that is capable of detoxifying

methotrexate. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthesis cassette for producing a payload that is capable of detoxifying methotrexate. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthesis cassette for producing a payload that is capable of detoxifying methotrexate. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthesis cassette for producing a payload that is capable of detoxifying methotrexate. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthesis cassette for producing a payload that is capable of detoxifying methotrexate. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptide of SEQ ID NO: 141 or a functional fragment thereof. [00886] In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthesis cassette for producing a payload that is capable of detoxifying irinotecan. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1.4-lactone (SAL) or biosynthesis cassette for producing a payload that is capable of detoxifying irinotecan. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthesis cassette for producing a payload that is capable of detoxifying irinotecan. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthesis cassette for producing a payload that is capable of detoxifying irinotecan. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding acetete or biosynthesis cassette for producing a payload that is capable of detoxifying irinotecan. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00887] In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding a short-chain fatty acid or biosynthesis cassette for producing a payload that is capable of detoxifying a NSAID. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthesis cassette for producing a payload that is capable of detoxifying a NSAID. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthesis cassette for producing a payload that is capable of detoxifying a NSAID. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding acetete or biosynthesis cassette for producing a payload that is capable of detoxifying a NSAID. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthesis cassette for producing a payload that is capable of detoxifying a NSAID. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof. In one embodiment, the NSAID is naproxen.

[00888] In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthesis cassette for producing a payload that is capable of detoxifying heavy metals. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthesis cassette for producing a payload that is capable of detoxifying heavy metals. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthesis cassette for producing a payload that is capable of detoxifying heavy metals. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthesis cassette for producing a payload that is capable of detoxifying heavy metals. In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthesis cassette for producing a payload that is capable of detoxifying heavy metals. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00889] In one embodiment, the genetically engineered bacteria comprises a nucleic acid encoding a biosynthesis cassette for producing a payload that is capable of detoxifying one or more environmental toxic molecules, such as antibiotics, anti-convulsants, mood stabilizers and sex hormones. In some embodiments the genetically engineered bacteria further comprises a nucleic acid encoding one of an effector molecule or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00890] In one embodiments the genetically engineered bacteria comprises a nucleic acid encoding IL-10 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-2 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-22 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-27 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-20 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-24 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-19 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IL-10 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding SOD or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding GLP2 or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding IFN-γ or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding TNF-a or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding 1L-1B or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding tryptophan or biosynthetic cassette for producing a payload that is capable of enhancing a gut barrier function and/or anti- inflammation.

[00891] In some embodiments the genetically engineered bacteria comprises a nucleic acid encoding one of an effector molecule or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with one of an effector molecule or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding

carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding

carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase Gi (CPD Gi) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00892] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding

carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding carboxypeptidase G ₂ (CPD G ₂ ) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptide of SEQ ID NO: 141 or a functional fragment thereof.

[00893] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D- saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D- saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D- saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding D-saccharic acid 1, 4-lactone (SAL) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00894] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or

glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10- hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or

glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10- hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or

glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10- hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or

glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl-10- hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or

glucuroniding 7-ethyl-10-hydroxycamptothecin (SN-38) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00895] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or

glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal antiinflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal antiinflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal antiinflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal antiinflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal antiinflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00896] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding naproxen or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00897] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a proton pump inhibitor or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00898] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator a heavy metal chelator or

biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a heavy metal chelator or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00899] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a plant phytochelatin or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof. [00900] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with butyrate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with propionate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with acetate or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short- chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short- chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one

embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short- chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a short-chained fatty acid or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00901] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding a butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding butyrate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof. [00902] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding propionate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

[00903] In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-22 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-27 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-20 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-24 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-19 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IL-10 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with SOD or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with GLP2 or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with IFN-γ or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with TNF-a or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with 1L-1B or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti-inflammation. In one embodiment the genetically engineered bacteria comprises a nucleic acid encoding acetate or biosynthetic cassette for producing a first payload that is capable of detoxifying a deleterious molecule, in combination with tryptophan or biosynthetic cassette for producing a second payload that is capable of enhancing a gut barrier function and/or anti- inflammation. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 35-44 or a functional fragment thereof. In one embodiment, the bacteria comprise a nucleic acid sequence that encodes the polypeptides of SEQ ID NOs: 45-94 or a functional fragment thereof.

RNAi, scFV, other Mechanisms

[00904] RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in plants and animals. RNAi is activated when microRNA (miRNA), double- stranded RNA (dsRNA), or short hairpin RNA (shRNA) is processed into short interfering RNA (siRNA) duplexes (Keates et al., 2008). RNAi can be "activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver shRNA to target cells" such as mammalian cells (Keates et al., 2008). In some embodiments, the genetically engineered bacteria of the invention induce RNAi-mediated gene silencing of one or more proinflammatory molecules in low-oxygen conditions. In some embodiments, the genetically engineered bacteria produce siRNA targeting TNF in low-oxygen conditions.

[00905] Single-chain variable fragments (scFv) are "widely used antibody fragments... produced in prokaryotes" (Frenzel et al., 2013). scFv lacks the constant domain of a traditional antibody and expresses the antigen-binding domain as a single peptide.

Bacteria such as Escherichia coli are capable of producing scFv that target pro-inflammatory cytokines, e.g., TNF (Hristodorov et al, 2014). In some embodiments, the genetically engineered bacteria of the invention express a binding protein for neutralizing one or more proinflammatory molecules in low-oxygen conditions. In some embodiments, the genetically engineered bacteria produce scFv targeting TNF in low-oxygen conditions. In some

embodiments, the genetically engineered bacteria produce both scFv and siRNA targeting one or more pro-inflammatory molecules in low-oxygen conditions (see, e.g., Xiao et al., 2014).

[00906] One of skill in the art would appreciate that additional genes and gene cassettes capable of producing enzymes capable of detoxifying a deleterious molecule, anti- inflammation and/or gut barrier function enhancer molecules are known in the art and may be expressed by the genetically engineered bacteria of the invention. In some embodiments, the gene or gene cassette for producing a therapeutic molecule also comprises additional transcription and translation elements, e.g. , a ribosome binding site, to enhance expression of the therapeutic molecule.

[00907] In some embodiments, the genetically engineered bacteria produce two or more enzymes capable of detoxifying a deleterious molecule, anti- inflammation and/or gut barrier function enhancer molecules. In certain embodiments, the two or more molecules behave synergistically to reduce gut inflammation and/or enhance gut barrier function. In some embodiments, the genetically engineered bacteria express at least one enzymes capable of detoxifying a deleterious molecule, at least one anti-inflammation molecule and at least one gut barrier function enhancer molecule. In certain embodiments, the genetically engineered bacteria express IL- 10 and GLP-2. In alternate embodiments, the genetically engineered bacteria express IL- 10 and butyrate.

[00908] In some embodiments, the genetically engineered bacteria are capable of producing IL-2, IL- 10, IL-22, IL-27, propionate, and butyrate. In some embodiments, the genetically engineered bacteria are capable of producing IL- 10, IL-27, GLP-2, and butyrate. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2, IL- 10, IL-22, SOD, butyrate, and propionate. In some embodiments, the genetically engineered bacteria are capable of GLP-2, IL-2, IL- 10, IL-22, IL-27, SOD, butyrate, and propionate. Any suitable combination of therapeutic molecules may be produced by the genetically engineered bacteria.

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Examples

[00910] The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.

[00911] An exemplary wild-type sequence of carboxypeptidase G ₂ (CPD G ₂ ) is shown below:

[00912] Fragments and modifications to those sequences may be designed according to methods known in the art. Bacteria are engineered to comprise a nucleic acid sequence that encodes the polypeptide of SEQ ID NO: 141 or a functional fragment thereof.

[00913] The CPD G ₂ gene is expressed under the control of each of the following promoters: a constitutive promoter, a tetracycline-inducible promoter with the tet repressor (TetR) expressed constitutively on a plasmid, or a FNR promoter selected from SEQ ID NOs: 1-16. As discussed herein, other promoters may be used.

[00914] The CPD G ₂ gene is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. The CPD G ₂ gene is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used. The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the CPD G ₂ construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting E. coli Nissle bacteria are genetically engineered to express CPD G ₂ and are capable of reducing

methotrexate-induced diarrhea and gastrointestinal toxicity.

Example 2: Constructs for producing butyrate

[00915] In some embodiments, the E. coli Nissle further comprise a gene cassette for producing butyrate. To facilitate the production of butyrate, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk; NCBI), as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 (Construct 1). The butyrate gene cassette is placed under control of of the following promoters: a constitutive promoter, a tetracycline-inducible promoter with the tet repressor (TetR) expressed constitutively on a plasmid, or a FNR promoter selected from SEQ ID NOs: 1-16. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

[00916] The butyrogenic gene cassette is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. The gene cassette is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used. The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the butyrogenic construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below.

[00917] The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co- oxidant. Because the genetically engineered bacteria are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from Treponema denticola, trans-2-enoynl-CoA reductase (ter), can functionally replace this three gene complex in an oxygen-independent manner. Therefore, a second construct capable of butyrate production in E. coli is created. Inverse PCR is used to amplify the entire sequence of Construct 1 outside of the bcd-etfA3-etfB3 region. The ter gene is codon optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool

(https://www.idtdna.com/CodonOpt), synthesized (Genewiz, Cambridge, MA), and cloned into this inverse PCR fragment using Gibson assembly to create Construct 2. The wild-type genomic sequences comprising the butryogenic genes are provided herein.

Example 3. Construction of Vectors for Producing Butyrate

[00918] To facilitate inducible production of butyrate in Escherichia coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk; NCBI; Table 36 and Table 37), as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 to create pLogic031 (bcd2-etfB3-etfA3-thiAl-hbd- crt2-pbt buk butyrate cassette, also referred to as bcd2-etfB3-etfA3 butyrate cassette, SEQ ID NO: 151).

[00919] Table 36 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes. Table 36. Exemplary Butyrate Cassette Sequences

[00920] The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA and may exhibit dependence on oxygen as a co-oxidant. Because the recombinant bacteria of the invention are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from Treponema denticola, trans-2-enoynl-CoA reductase (ter, Table 36 and Table 37), can functionally replace this three gene complex in an oxygen- independent manner. Therefore, a second butyrate gene cassette in which the ter gene replaces the bcd2-etfA3-etfB3 genes of the first butyrate cassette is synthesized (Genewiz, Cambridge, MA). The ter gene is codon- optimized for E. coli codon usage using Integrated DNA Technologies online codon

optimization tool (https://www.idtdna.com/CodonOpt). The second butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 to create pLogic046 (ter-thiAl-hbd- crt2-pbt buk butyrate cassette, also referred to herein as ter butyrate cassette or pbt buk butyrate cassette, SEQ ID NO: 152).

[00921] In a third butyrate gene cassette, the pbt and buk genes are replaced with tesB (SEQ ID NO: 150). TesB is a thioesterase found in E. Coli that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk (see, e.g., FIG. 5 and Table 36 and Table 37). The third butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 to create pLOGIC046-delta pbt.buk/tesB+ (ter-thiAl-hbd- crt2-tesb butyrate cassette, also referred to herein as tesB butyrate cassette, SEQ ID NO: 153). Table 37 lists non-limiting examples for sequences of the three cassettes.

Table 37. Butyrate Cassette Sequences

[00922] In certain constructs, the butyrate gene cassette (e.g., bcd2-etfB3-etfA3- thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiAl-hbd- crt2-tesb butyrate cassette (pLOGIC046- delta pbt.buk/tesB+)) is placed under the control of an RNS -responsive regulatory region, e.g., norB. In some embodiments, the butyrate gene cassette is placed under the control of an RNS- responsive regulatory region, e.g., norB. and the bacteria further comprises a gene encoding a corresponding RNS -responsive transcription factor, e.g., nsrR (see, e.g., Table 38 and Table 39 and SEQ ID NO: 156).

[00923] Table 38 depicts the nucleic acid sequence of an exemplary RNS- regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct; SEQ ID NO: 154). The sequence encoding NsrR is underlined and bolded, and the NsrR binding site, i.e., a regulatory region of norB is |boxed|. Table 39 depicts the nucleic acid sequence of an exemplary RNS- regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrogenic gene cassette (pLogic046-nsrR-norB-butyrate construct; SEQ ID NO: 155). The sequence encoding NsrR is underlined and bolded, and the NsrR binding site, i.e., a regulatory region of norB is |boxed|

[00924] Table 40 (SEQ ID NO: 156) depicts the nucleic acid sequence of an exemplary RNS-regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrogenic gene cassette (pLOGIC046-delta pbt.buk/tesB+-nsrR-norB-butyrate construct (SEQ ID NO: 156). [00925] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 154, 155, 156, or a functional fragment thereof.

[00926] In certain constructs, the butyrate gene cassette(e.g., bcd2-etfB3-etfA3- thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiAl-hbd- crt2-tesb butyrate cassette (pLOGIC046- delta pbt.buk/tesB+)) is placed under the control of an ROS -responsive regulatory region, e.g., oxyS. In certain constructs, the butyrate gene cassette {e.g., bcd2-etfB3-etfA3-thiAl-hbd- crt2- pbt buk butyrate cassette (pLogic031), and/or ter-thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiAl-hbd- crt2-tesb butyrate cassette (pLOGIC046-delta

pbt.buk/tesB+)) is placed under the control of an ROS -responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS -responsive transcription factor, e.g., oxyR.

[00927] Nucleic acid sequences of exemplary ROS-regulated constructs comprising an oxyS promoter are shown in Table 41 and Table 42 and Table 44.. The nucleic acid sequence of an exemplary construct encoding OxyR is shown in Table 43. Table 41 depicts the nucleic acid sequence of an exemplary ROS-regulated construct comprising an oxyS promoter and a butyrogenic gene cassette (pLogic031-oxyS -butyrate construct; SEQ ID NO: 157). Table 42 depicts the nucleic acid sequence of an exemplary ROS-regulated construct comprising an oxyS promoter and a butyrogenic gene cassette (pLogic046-oxyS -butyrate construct; SEQ ID NO: 158). Table 43 depicts the nucleic acid sequence of an exemplary construct encoding OxyR (pZA22-oxyR construct; SEQ ID NO: 159). Table 44 depicts the nucleic acid sequence of an exemplary ROS-regulated construct comprising an oxyS promoter and a butyrogenic gene cassette (pLOGIC046-delta pbt.buk/tesB+ -oxyS -butyrate construct; SEQ ID NO: 160).

[00928] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 157, 158, 159, or 160, or a functional fragment thereof.

[00929] In some embodiments, the butyrate gene cassette (e.g. , bcd2-etfB3- etfA3-thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiAl-hbd- crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiAl-hbd- crt2-tesb butyrate cassette

(pLOGIC046-delta pbt.buk/tesB+)) is placed under the control of a FNR regulatory region selected from Table 3 or 4 and SEQ ID NOs: 1-17. In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

Example 4. Construction of vectors for overproducing butyrate using an inducible tet promoter- butyrate circuit

[00930] To facilitate inducible production of butyrate in Escherichia coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiAl, hbd, crt2, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 to create pLogic031. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter.

[00931] The gene products of bcd2-etfA3-etfB3 form a complex that convert crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co-oxidant. For reasons described in Example 3, a second plasmid was generated, in which bcd2-etfA3- etfB3 was replaced with (trans-2-enoynl-CoA reductase; ter from Treponema denticola capable of butyrate production in E. coli. Inverse PCR was used to amplify the entire sequence of pLogic031 outside of the bcd-etfA3-etfB3 region. The ter gene was codon optimized for E. coli codon usage using Integrated DNA technologies online codon optimization tool, synthesized (Genewiz, Cambridge, MA), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic046.

[00932] A third butyrate gene cassette was further genereated, in which the pbt and buk genes were replaced with tesB (SEQ ID NO: 150). TesB is a thioesterase found in E. Coli that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk (see Fig. 5). The third butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 to create pLOGIC046- delta pbt.buk/tesB+ (ter-thiAl-hbd- crt2-tesb butyrate cassette, also referred to herein as tesB butyrate cassette).

[00933] As synthesized, the all three butyrate gene cassettes were placed under control of a tetracycline-inducible promoter, with the tet repressor (tetR) expressed

constitutively, divergent from the tet-inducible synthetic butyrate operon.

[00934] Nucleic acid sequences of tetracycline-regulated constructs comprising a tet promoter are shown in Table 45 and Table 46 and Table 47. Table 45 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct comprising a tet promoter and a butyrogenic gene cassette (pLogic031-tet-butyrate construct; SEQ ID NO: 161). The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are |boxed|. Table 46 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct comprising a tet promoter and a butyrogenic gene cassette (pLogic046-tet-butyrate construct; SEQ ID NO: 161). The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are boxed

[00935] Table 47 depicts the nucleic acid sequence of an exemplary tetracycline- regulated construct (pLOGIC046-delta pbt.buk/tesB+- tet-butyrate construct) comprising a reverse complement of the tetR repressor (underlined), an intergenic region containing divergent promoters controlling tetR and the butyrate operon and their respective RBS (bold), and the butyrate genes separated by RBS.

[00936] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 161, 162, or 163, or a functional fragment thereof.

Example 5. Production of Butyrate in Recombinant E. coli using tet-inducible promoter

[00937] Production of butyrate was assessed in E. coli Nissle strains containing butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. The tet-inducible cassettes tested include (1) tet-butyrate cassette comprising all eight genes (pLOGIC031); (2) tet-butyrate cassette in which the ter is substituted

(pLOGIC046) and (3) tet-butyarte cassette in which tesB is substituted in place of pbt and buk genes.

[00938] All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells were grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ ). One mL culture aliquots were prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

[00939] FIG. 6 depicts bar graphs of butyrate production using the different butyrate-producing circuits shown in FIG. 5.

[00940] FIG. 6 shows butyrate production in strains pLOGIC031 and

pLOGIC046 in the presence and absence of oxygen, in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl Co A).

Example 6. Tet-driven and RNS driven in vitro Butyrate Production in

Recombinant E. coli

[00941] All incubations were performed at 37°C. Lysogeny broth (LB)-grown overnight cultures of E. coli Nissle transformed with pLogic031 or pLogic046 were

subcultured 1: 100 into lOmL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2h, at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of lOOng/mL to induce expression the butyrate operon from pLogic031 or pLogic046. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture

supernatant was then analyzed at indicated time points ((0 up to 24 hours, as shown in FIG. 21) to assess levels of butyrate production by LC-MS. As seen in FIG. 21 butyrate production is greater in the strain comprising the pLogic046 construct than the strain comprising the pLogic031 construct.

[00942] Production of butyrate was also assessed in E. coli Nissle strains containing the butyrate cassettes driven by an RNS promoter described above (pLogic031- nsrR-norB -butyrate operon construct and pLogic046-nsrR-norB-butyrate operon construct) in order to determine the effect of nitrogen on butyrate production. Overnight bacterial cultures were diluted 1: 100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine- nitric oxide adduct) was added to cultures at a final concentration of 0.3mM to induce expression from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points (0 up to 24 hours, as shown in FIG. 22) to assess levels of butyrate production. As seen in FIG. 22, genetically engineered Nissle comprising pLogic031- nsrR-norB -butyrate operon construct) or (pLogic046-nsrR-norB-butyrate operon construct) produced significantly more butyrate as compared to wild-type Nissle.

Example 7. In vitro Production of butyrate in Recombinant E. coli using an Inducible tet Promoter Butyrate Circuit

[00943] NuoB is a protein complex involved in the oxidation of NADH during respiratory growth (form of growth requiring electron transport). Preventing the coupling of NADH oxidation to electron transport allows an increase in the amount of NADH being used to support butyrate production. To test whether Preventing the coupling of NADH oxidation to electron transport would allow increased butyrate production, NuoB mutants having NuoB deletion were obtained.

[00944] All incubations were performed at 37°C. Lysogeny broth (LB)-grown overnight cultures of E. coli strains DH5a and Nissle containing pLogic031 or pLogic046 were subcultured 1: 100 into lOmL of M9 minimal medium containing 0.2% glucose and grown shaking (200 rpm) for 2h, at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of lOOng/mL to induce expression the butyrate operon from pLogic031 or pLogic046. Cultures were incubated either shaking in flasks (+0 ₂ ) or in the anaerobic chamber (-0 ₂ ) and samples were removed, and butyrate was quantitated at 2, 4, and 24hr via LC-MS. See FIG. 13, which depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. FIG. 13 shows the BW25113 strain of E. Coli, which is a common cloning strain and the background of the KEIO collection of E. Coli mutants. FIG. 13 shows that compared with wild-type Nissle, deletion of NuoB results in greater production of butyrate.

Example 8. Production of Butyrate in Recombinant E. coli

[00945] In vitro production of butyrate under the control of a tetracycline promoter was compared between (1) Butyrate gene cassette (pLOGIC046- ter-thiAl-hbd- crt2- pbt buk butyrate) and (2) butyrate cassette in which the pbt and buk genes were placed with tesB (pLOGIC046-deltapbt-buk/tesB+-butyrate; SEQ ID NO: 167).

[00946] Overnight bacterial cultures were diluted 1: 100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, anhydrous tetracycline (ATC) was added to cultures at a final concentration of 100 ng/mL to induce expression of butyrate genes from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. As shown in FIG. 6, replacement of pbt and buk with tesB leads to greater levels of butyrate production.

Example 9. Construction of vectors for overproducing butyrate (FNR driven)

[00947] The three butyrate cassettes decribed in Example 3 (see, e.g., Table 37,

SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153) are placed under the control of a FNR regulatory region selected from (SEQ ID NO: 1 through SEQ ID NO: 17) (Table 3 and

Table 4) In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. In certain embodiments, a ydfZ promoter was used. In other embodiments, a FNRS promoter is used.

Example 10. FNR and RNS driven in vitro Production of Butyrate in

Recombinant E. coli

[00948] Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above driven by an FNR promoter in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 niL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ ). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

[00949] In an alternate embodiment, production of butyrate is assessed in E. coli

Nissle strains containing the butyrate cassettes described above driven by an RNS promoter in order to determine the effect of nitrogen on butyrate production. Overnight bacterial cultures are diluted 1: 100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine- nitric oxide adduct) is added to cultures at a final concentration of 0.3mM to induce expression from plasmid. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at indicated time points to assess levels of butyrate production.

Example 11. Production of Butyrate in Recombinant E. coli

[00950] The effect of oxygen and glucose on FNR promoter driven butyrate production was compared between E. coli Nissle strains SYN501( comprises pSClOl PydfZ- ter butyrate plasmid, i.e., (ter-thiAl-hbd-crt2-pbt-buk genes under the control of a ydfZ promoter) SYN-UCD500 (comprises pSClOl PydfZ-bcd butyrate plasmid, i.e, bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk under control of the ydfZ promoter) and SYN-UCD506 (comprises pSClOl nirB-bcd butyrate plasmid, i.e., i.e, bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk under control of the nirB promoter.

[00951] All incubations were performed at 37° C. Cultures of E. coli Nissle strains transformed with the butyrate cassettes were grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells were grown with shaking (250 rpm) for 4-6 h and incubated anaerobically in a Coy anaerobic chamber

(supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ ) for 4 hours. Cells were washed and resuspended in minimal media w/ 0.5% glucose and incubated microaerobically to monitor butyrate production over time. One aliquot was removed at each time point (2, 8, and 24 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment. As seen in FIG. 9B, SYN-501 led to significant butyrate production under anaerobic conditions..

[00952] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 164, 165, 166, or 61, or a functional fragment thereof.

ID ID

164

Example 12. Production of Butyrate in Recombinant E. coli

[00953] The effect of oxygen and glucose on butyrate production was assessed in E. coli Nissle strains using a butyrate cassette driven by a FNR promoter (ter-thiAl-hbd- crt2-pbt-buk genes under the control of a ydfZ promoter). [00954] All incubations were performed at 37° C. Cultures of E. coli strains

DH5a and Nissle transformed with the butyrate cassettes were grown overnight in LB and then diluted 1:200 into 4 mL of LB containing no glucose or RCM medium containing 0.5% glucose. The cells were grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ ). One mL culture aliquots were prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube was removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

[00955] FIG. 9C depicts butyrate production in strains comprising an FNR- butyrate cassette (having the ter substitution) in the presence/absence of glucose and oxygen and shows that bacteria need both glucose and anaerobic conditions for butyrate production from the FNR promoter. Cells were grown aerobically or anaerobically in media containg no glucose (LB) or in media containing glucose at 0.5% (RMC). Culture samples were taken at indicaed time pints and supernatant fractions were assessed for butyrate concentration using LC-MS. These data show that SYN501 requires glucose for butyrate production and that in the presence of glucose butyrate production can be enhanced under anaerobic conditions when under the control of the anaerobic FNR-regulated ydfZ promoter.

Example 13. Comparison of in vitro butyrate production efficacy of chromosomal insertion and plasmid-bearing engineered bacterial strains

[00956] The in vitro butyrate production efficacy of engineered bacterial strains harboring a chromosomal insertion of a butyrate cassette was compared to a strain bearing a butyrate cassette on a plasmid. SYN1001 and SYN1002 harbor a chromosomal insertion between the agal/rsml locus of a butyrate cassette (either ter- tesB or ter- pbt-buk, respectively) driven by an fnr inducible promoter. These strains were compared side by side with the low copy plasmid strain SYN501 (Logicl56 (pSClOl PydfZ-ter ->pbt-buk butyrate plasmid) also driven by an fnr inducible promoter. Butyrate levels in the media were measured at 4 and 24 hours post anaerobic induction.

[00957] Briefly, 3ml LB was inoculated with bacteria from frozen glycerol stocks. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1: 100 dilution into 10ml LB (containing antibiotics) in a 125ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X10 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At indicated times (4 and 24h), 120 ul cells were removed and pelleted at 14,000rpm for lmin, and lOOul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate concentrations (as described in other examples). Results are depicted in FIG. 12, and show that SYN1001 and SYN1002 give comparable butyrate production to the plasmid strain SYN501.

[00958] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 168, 169, 170, or 171, or a functional fragment thereof.

actataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttata gcattag

Example 14. Assessment of intestinal butyrate levels in response to SYN501 administration in mice

[00959] To determine efficacy of butyrate production by the genetically engineered bacteria in vivo, the levels of butyrate upon administration of SYN501 (Logicl56 (pSClOl PydfZ-ter ->pbt-buk butyrate plasmid)) to C57BL6 mice was first assessed in the feces. Water containing 100 mM butyrate was used as a control.

[00960] On day 1, C57BL6 mice (24 total animals) were weighed and randomized into 4 groups; Group 1: H20 control (n=6); Group 2-100 mM butyrate (n=6); Group 3 -streptomycin resistant Nissle (n=6); Group 4-SYN501 (n=6). Mice were either gavaged with 100 ul streptomycin resistant Nissle or SYN501, and group 2 was changed to H20(+)100 mM butyrate at a dose of lOelO cells/lOOul. On days 2-4, mice were weighted and Groups 3 and 4 were gavaged in the AM and the PM with streptomycin resistant Nissle or SYN501. On day 5, mice were weighed and Groups 3 and 4 were gavaged in the am with streptomycin resistant Nissle or SYN501, and feces was collected and butyrate concentrations determined as described in Example 23. Results are depicted in FIG. 10. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM butyrate.

[00961] Next the effects of SYN501 on levels of butyrate in the cecum, cecal effluent, large intestine, and large intestine effluent are assessed. Because baseline

concentrations of butyrate are high in these compartments, an antibiotic treatment is administered in advance to clear out the bacteria responsible for butyrate production in the intestine. As a result, smaller differences in butyrate levels can be more accurately observed and measured. Water containing 100 mM butyrate is used as a control. [00962] During week 1 of the study, animals are treated with an antibiotic cocktail in the drinking water to reduce the baseline levels of resident microflora. The antibiotic cocktail is composed of ABX-ampicillin, vancomycin, neomycin, and metronidazole. During week 2 animals are orally administered 100 ul of streptomycin resistant Nissle or engineered strain SYN501 twice a day for five days (at a dose of lOelO cells/lOOul).

[00963] On day 1, C57BL6 (Female, 8 weeks) are separated into four groups as follows: Group 1: H20 control (n=10); Group 2: 100 mM butyrate (n=10); Group 3:

streptomycin resistant Nissle (n=10); Group 4: SYN501 (n=10). Animals are weighed and feces is collected from the animals (T=0-time point). Animals are changed to H20 (+) antibiotic cocktail. On day 5, animals are weighed and feces is collected (time point T=5d). The H20 (+) antibiotic cocktail bottles are changed. On day 8, the mice are weighed and feces is collected. Mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. The water in all cages is changed to water without antibiotic.

Group 2 is provided with 100 mM butyrate in H20. On days 9-11, mice are weighed, and mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. On day 12, mice are gavaged with streptomycin resistant Nissle or SYN501 in the AM, and 4 hours post dose, blood is harvested, and cecal and large intestinal contents, and tissue, and feces are collected and processed for analysis.

Example 15. Comparison of Butyrate production levels between the genetically engineered bacteria encoding a butyrate cassette and selected Clostridia strains

[00964] The efficacy of pbutyrate production in SYN501 (pSClOl PydfZ-ter -

>pbt-buk butyrate plasmid) was compared to CBM588 (Clostridia butyricum MIYARISAN, a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain).

[00965] Briefly, overnight cultures of SYN501 were diluted 1: 100 were grown in

RCM (Reinforced Clostridial Media, which is similar to LB but contains 0.5% glucose) at 37 C with shaking for 2 hours, then either moved into the anaerobic chamber or left aerobically shaking. Clostridial strains were only grown anaerobically. At indicated times (2, 8, 24, and 48h), 120 ul cells were removed and pelleted at 14,000rpm for lmin, and lOOul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate concentrations (as described in Example 16). Results are depicted in FIG. 11, and show that SYN501 produces butyrate levels comparable to Clostridium spp. in RCM media. Example 16. Quantification of Butyrate by LC-MS/MS

[00966] To obtain the butyrate measurements in Example 23 a LC-MS/MS protocol for butyrate quantification was used.

Sample preparation

[00967] First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 μ g/mL sodium butyrate standards were prepared in water. Then, 10 μ L of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90 μ L of 67% ACN (60uL ACN+30uL water per reaction) with 4ug/mL of butyrate-d7 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 20 μ L of diluted samples were added to 180 μ L of a buffer containing lOmM MES pH4.5, 20mM EDC (N-iB-Dimethylaminopropyfl-N' -ethylcarbodiimide), and 20mM TFEA (2,2,2- trifluroethylamine). The plate was again heat- sealed and mixed well, and samples were incubated at room temperature for 1 hour.

LC-MS/MS method

[00968] Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 50 and Table 51. Tandem Mass Spectrometry details are found in Table 52.

Example 17. Quantification of Butyrate in feces by LC-MS/MS

Sample preparation

[00969] Fresh 1000, 500, 250, 100, 20, 4 and 0.8μg/mL sodium butyrate standards were prepared in water. Single fecal pellets were ground in lOOuL water and centrifuged at 15,000 rpm for 5min at 4°C. ΙΟμL of the sample (fecal supernatant and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90μL of the derivatizing solution containing 50mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylpho spine in acetonitrile with 5ug/mL of butyrate-d ₇ were added to each sample. The plate was heat-sealed and incubated at 60°C for lhr. The plate was then centrifuged at 4,000rpm for 5min and 20μL of the derivatized samples mixed to 180μL of 22% acetonitrile with 0.1% formic acid.

LC-MS/MS method

[00970] Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 53 and Table 54. Tandem Mass Spectrometry details are found in Table 54. Table 53. HPLC Details

Table 54. HPLC Method

Table 55. Tandem Mass Spectrometry Details

Example 18. Lambda red recombination

[00971] Lambda red recombination is used to make chromosomal modifications, e.g., to express CPD G ₂ in E. coli Nissle. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of E. coli. A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1: 100 in 5 mL of LB media and grown until it reaches an OD ₆ oo of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.

[00972] DNA sequences comprising the desired CPD G ₂ sequences shown above were ordered from a gene synthesis company. The lambda enzymes are used to insert this construct into the genome of E. coli Nissle through homologous recombination. The construct is inserted into a specific site in the genome of E. coli Nissle based on its DNA sequence. In some embodiments, the construct is in the E. coli Nissle genome at the malP/T site (Fig. 24). To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified, and includes approximately 50 bases on either side of the sequence. The homologous sequences are ordered as part of the synthesized gene. Alternatively, the homologous sequences may be added by PCR. The construct includes an antibiotic resistance marker that may be removed by recombination. The resulting construct comprises

approximately 50 bases of homology upstream, a kanamycin resistance marker that can be removed by recombination, the CPD G ₂ gene, and approximately 50 bases of homology downstream.

Example 19. Transforming E. coli Nissle

[00973] The CPD G ₂ construct above is transformed into E. coli Nissle comprising pKD46. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture is diluted 1: 100 in 5 mL of LB media containing ampicillin and grown until it reaches an OD ₆ oo of 0.1. 0.05 mL of 100X L-arabinose stock solution is added to induce pKD46 lambda red expression. The culture is grown until it reaches an OD ₆ oo of 0.4-0.6. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 0.5 μg of the mutated ARG box construct is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing kanamycin and incubated overnight.

Example 20. Verifying mutants

[00974] The presence of the CPD G ₂ is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 μΐ of cold ddH ₂ 0 by pipetting up and down. 3 μΐ of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C overnight. A PCR master mix is made using 5 μΐ of 10X PCR buffer, 0.6 μΐ of 10 mM dNTPs, 0.4 μΐ of 50 mM Mg ₂ S0 ₄ , 6.0 μΐ of 10X enhancer, and 3.0 μΐ of ddH ₂ 0 (15 μΐ of master mix per PCR reaction). A 10 μΜ primer mix is made by mixing 2 μϊ _^ of primers unique to the CPD G ₂ construct (100 μΜ stock) into 16 μΐ _^ of ddH ₂ 0. For each 20 μΐ reaction, 15μί of the PCR master mix, 2.0 μΐ _^ of the colony suspension

(template), 2.0 μΐ _^ of the primer mix, and 1.0 μΐ _^ of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C at 5:00 min., 2) 94° C at 0: 15 min., 3) 55° C at 0:30 min., 4) 68° C at 2:00 min., 5) 68° C at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 μΐ _^ of each amplicon and 2.5 μΐ _^ 5X dye. The PCR product only forms if the mutation has inserted into the genome.

Example 21. Removing selection marker

[00975] The antibiotic resistance gene is removed with pCP20. Each strain with the CPD G ₂ is grown in LB media containing antibiotics at 37° C until it reaches an OD ₆ oo of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pCP20 plasmid DNA is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette was placed into the sample chamber, and the electric pulse was applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1-3 hrs. The cells are spread out on an LB plate containing kanamycin and incubated overnight. Colonies that do not grow to a sufficient OD ₆ oo overnight are further incubated for an additional 24 hrs. 200 μΐ _^ of cells are spread on ampicillin plates, 200 μΐ _^ of cells are spread on kanamycin plates, and both are grown at 37° C overnight. The ampicillin plate contains cells with pCP20. The kanamycin plate provides an indication of how many cells survived the electroporation. Transformants from the ampicillin plate are purified non- selectively at 43° C and allowed to grow overnight.

Example 22. Verifying transformants

[00976] The purified transformants are tested for sensitivity to ampicillin and kanamycin. A colony from the plate grown at 43° C is picked and and resuspended in 10 μΐ _^ of LB media. 3 μΐ _^ of the cell suspension is pipetted onto each of three plates: 1) an LB plate with kanamycin incubated at 37° C, which tests for the presence or absence of the KanR gene in the genome of the host strain; 2) an LB plate with ampicillin incubated at 30° C, which tests for the presence or absence of the AmpR gene from the pCP20 plasmid; and 3) an LB plate without antibiotic incubated at 37° C. If no growth is observed on the kanamycin or ampicillin plates for a particular colony, then both the KanR gene and the pCP20 plasmid were lost, and the colony is saved for further analysis. The saved colonies are restreaked onto an LB plate to obtain single colonies and grown overnight at 37° C. The presence of the CPD G ₂ is confirmed by sequencing the genome.

Example 23. Production of butyrate in genetically engineered E. coli

[00977] Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1 :200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h, and the inducible constructs are induced as follows: (1) bacteria comprising a butyrate gene cassette driven by a FNR- inducible promoter are induced in LB at 37C for up to 4 hours in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ , and 20mM nitrate) at 37° C; (2) bacteria comprising a butyrate gene cassette driven by a tetracycline-inducible promoter are induced with

anhydrotetracycline (lOOng/mL); (3) bacteria comprising a butyrate gene cassette driven by a arabinose-inducible promoter are inducedwith 1% arabinose in media lacking glucose. One mL culture aliquots are prepared in 1.5 mL capped tubes. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains.

Example 24. Production of CPD G ₂ in genetically engineered E. coli

[00978] In some embodiments, the genetically engineered bacteria described above are grown overnight in LB at 37C with shaking. The bacteria are diluted 1 : 100 in 5mL LB and grown at 37C with shaking for 1.5 hr. The bacteria cultures are induced as follows: (1) bacteria comprising FNR- inducible CPD G ₂ are induced in LB at 37C for up to 4 hours in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ , and 20mM nitrate) at 37° C; (2) bacteria comprising tetracycline-inducible CPD G ₂ are induced with anhydrotetracycline (lOOng/mL); (3) bacteria comprising arabinose-inducible CPD G ₂ are inducedwith 1% arabinose in media lacking glucose. After induction, bacterial cells are removed from the incubator and spun down at maximum speed for 5 minutes. The cells are resuspended in 1 mL M9 glucose, and the OD ₆ oo is measured. Cells are diluted until the OD ₆ oo is between 0.6-0.8. Resuspended cells in M9 glucose media are grown aerobically with shaking at 37C. 100 uL of the cell resuspension is removed and the OD ₆ oo is measured at time = 0. A 100 uL aliquot is frozen at -20C in a round-bottom 96-well plate for mass spectrometry analysis (LC-MS/MS). At each subsequent time point, 100 uL of the cell suspension is removed and the OD ₆ oo is measured; a 100 uL aliquot is frozen at -20C in a round-bottom 96- well plate for mass spectrometry analysis. Samples are analyzed for CPD G ₂ levels. At each time point, normalized concentrations as determined by mass spectrometry vs. OD ₆ oo are used to determine the rate of CPD G ₂ production per cell per unit time.

[00979] In some embodiments, the genetically engineered bacteria described above are streaked from glycerol stocks for single colonies on agar. A colony is picked and grown in 3 mL LB for four hours or overnight, then centrifuged for 5 min. at 2,500 rcf. The cultures are washed in M9 media with 0.5% glucose. The cultures are resuspended in 3 mL of M9 media with 0.5% glucose, and the OD ₆ oo is measured. The cultures are diluted in M9 media with 0.5% glucose, with or without ATC (100 ng/mL), with or without 20 mM glutamine, so that all of the OD ₆ oo are between 0.4 and 0.5. A 0.5 mL aliquot of each sample is removed, centrifuged for 5 min. at 14,000 rpm, and the supernatant is removed and saved. The supernatant is frozen at -80° C, and the cell pellets are frozen at -80° C (t=0). The remaining cells are grown with shaking (250 rpm) for 4-6 hrs and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , 5%H ₂ ) at 37° C. One 0.5 mL aliquot is removed from each sample every two hours and the OD ₆ oo is measured. The aliquots are centrifuged for 5 min. at 14,000 rpm, and the supernatant is removed. The supernatant is frozen at -80° C, and the cell pellets are frozen at -80° C (t=2, 4, and 6 hours). The samples are placed on ice, and CPD G ₂ levels are determined using mass spectrometry.

Example 25. Methotrexate toxicity in vivo

[00980] BDFi male mice are used to assay methotrexate-induced diarrhea and gastrointestinal toxicity (Chabner et al., 1972). Methotrexate is injected intraperito nearly at a dose of 350 or 1,000 mg/kg. The genetically engineered bacteria described above are grown overnight in LB. Bacteria are then diluted 1: 100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters is administered by oral gavage to mice daily for 1-7 days. Levels of blood methotrexate and metabolites are measured by HPLC, immunoassay, and/or enzyme inhibition assay. After bacterial treatment, gastrointestinal damage is scored in live mice using endoscopy.

Endoscopic damage score is determined by assessing colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. Mice are sacrificed and colonic tissues are isolated. Colonic sections are fixed and scored for damage. Colonic tissue is homogenized and measurements are made for myeloperoxidase activity using an enzymatic assay kit. As described herein, other models of chemotherapy-induced diarrhea may be used, e.g., lapatinib-induced diarrhea (Bowen et al., 2014).

Example 26. Generation of DeltaThyA

[00981] An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.

[00982] A thyAr. cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a lOOum concentration are found in Table 56. Table 56. Primer Sequences

[00983] For the first PCR round, 4x50ul PCR reactions containing lng pKD3 as template, 25ul 2xphusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions: stepl: 98c for 30s step2: 98c for 10s step3: 55c for 15s step4: 72c for 20s repeat step 2-4 for 30 cycles

step5: 72c for 5min

[00984] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm

PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30ul nuclease free water.

[00985] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[00986] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. {thyA auxotrophs will only grow in media supplemented with thy 3mM).

[00987] Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. lmL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in lmL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for Ihours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and lOOug/ml carbenicillin and grown at 30°C for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42°C.

[00988] To test the colony-purified transformants, a colony was picked from the

42°C plate with a pipette tip and resuspended in ΙΟμL LB. μL of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.

Example 27. Nissle residence

[00989] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non- limiting example using a streptomycin- resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.

[00990] C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation (i.e., day 0), streptomycin-resistant Nissle (SYN-UCD103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 57. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

Table 57: CFU administered via oral gavage

[00991] On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs.

1-6; Table 58). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

[00992] Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 58.

[00993] Fig. 26 depicts a graph of Nissle residence in vivo. Streptomycin- resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

Table 58. Nissle residence in vivo

Example 28. Intestinal Residence and Survival of Bacterial Strains in vivo

[00994] Localization and intestinal residence time of streptomycin resistant

Nissle, FIG. 26, was determined. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.

[00995] Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 10 ¹⁰ CFU/mL. Mice (C57BL6/J, 10- 12 weeks old) were gavaged with 100 μL of bacteria (approximately 10 ⁹ CFU). Drinking water for the mice was changed to contain 0.1 mg/mL anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post-gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.

[00996] In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37°C overnight, and colonies were counted. The amount of bacteria and residence time in each compartment is shown in Fig. 26.

Example 29. FNR promoter activity

[00997] In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 3. The nucleotide sequences of these constructs are shown in Tables 59-66 (SEQ ID NOs 178-185). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity. Alternatively, beta-galactosidase may be used as a reporter, exemplary results are shown in Fig. 16.

[00998] Table 59 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf _m i (SEQ ID NO: 178). The construct comprises a translational fusion of the Nissle nirBl gene and the lacZ gene, in which the translational fusions are fused in frame to the 8 ^th codon of the lacZ coding region. The Pfnri sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[00999] Table 60 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf _m 2 (SEQ ID NO: 179). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8 ^th codon of the lacZ coding region. The Pf _m 2 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[001000] Table 61 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf _m 3 (SEQ ID NO: 180). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The Pf _m 3 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[001001] Table 62 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P _fm 4 (SEQ ID NO: 181). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The P _fm 4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[001002] Table 63 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS (SEQ ID NO: 182). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrSl, fused to lacZ. The P _fms sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[001003] Table 64 shows the nucleotide sequence of an exemplary construct comprising a gene encoding PAL3, and an exemplary FNR promoter, P _fm 3 (SEQ ID NO: 183). The construct comprises a transcriptional fusion of the Nissle nirB gene and the PAL3 gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The P _fm 3 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The PAL3 sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[001004] Table 65 shows the nucleotide sequences of an exemplary construct comprising a gene encoding PAL3, and an exemplary FNR promoter, P _fm 4 (SEQ ID NO: 184). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the PAL3 gene. The P _fm 4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The PAL3 sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[001005] Table 66 shows the nucleotide sequences of an exemplary construct comprising a gene encoding PAL3, and an exemplary FNR promoter, P _fms (SEQ ID NO: 185). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrSl, fused to PAL3. The Pfnrs sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The PAL3 sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 59

[001006] Each of the plasmids was transformed into E. coli Nissle, as described above. Cultures of transformed E. coli Nissle were grown overnight and then diluted 1:200 in LB. The cells were grown with shaking at 250 rpm either aerobically or anaerobically in a Coy anaerobic chamber supplied with 90% N ₂ , 5% C0 ₂ , and 5% H ₂ . After 4-6 hrs of incubation, samples were collected, and promoter activity was analyzed by performing β-galactosidase assays (Miller, 1972). As shown in Fig. 16, the activities of the FNR promoters were greatly enhanced under anaerobic conditions compared to aerobic conditions.

Example 30. Nitric oxide-inducible reporter constructs

[001007] ATC and nitric oxide-inducible reporter constructs were synthesized

(Genewiz, Cambridge, MA). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations; promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and

RBS are boxed.

[001008] These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600= -0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half- life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units.

[001009] Dot blots comprising NO-GFP constructs E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1: 100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS- treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

[001010] Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1: 100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It was observed that NsrR-regulated promoters were induced in DSS -treated mice, but not in untreated mice.

Example 31. Measuring the activity of an FNR promoter

[001011] To determine the kinetics of FNR promoter-driven gene expression, E. coli strains harboring a low-copy fnrS-lacZ fusion gene (Fig. 17A) were grown aerobically with shaking at 250 rpm. Cultures were split after 1 hr., and then incubated either aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N ₂ , 5% C0 ₂ , and 5%H ₂ ) at 37 °C. Promoter activity was measured as a function of β-galactosidase activity using a standard colorimetric assay (Miller, 1972). Fig. 17B demonstrates that the fnrS promoter begins to drive high-level gene expression within 1 hr. under anaerobic conditions. Growth curves of bacterial cell cultures expressing lacZ are shown in Fig. 17C, both in the presence and absence of oxygen.