CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Serial No. 63/412,685, filed on October 3, 2022, the entire disclosure of which is incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0002] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 32.0 kilobytes ACII (xml) file named “85007-395554_SL.xml,” created on October 3, 2023.

BACKGROUND OF THE INVENTION

[0003] Uric acid is formed in the liver, intestines, muscles, and kidneys as a product of purine catabolism. This product remains in the body until approximately two-thirds of it is excreted via kidneys, and the remaining one third by way of the intestinal tract.

[0004] Most mammals can oxidize uric acid into allantoin using urate oxidase. Allantoin is highly soluble in water and can readily be excreted from kidneys. Unfortunately, humans do not express urate oxidase. Hence, overproduction or inefficient excretion of uric acid causes high concentrations of this compound in blood serum, a condition known as hyperuricemia. Hyperuricemia generally refers to higher serum uric acid levels, for example those exceeding 360 pM for women and 420 pM for men.

[0005] Despite its powerful antioxidant properties that counteract the effects of oxidative damage due to atherosclerosis and aging, chronic high concentrations of uric acid have adverse effects on human health. The prevalence of this disease is substantial among adults in the United States, affecting approximately 8.3 million individuals. Hyperuricemia is often associated with diseases, such as gout, metabolic syndrome, hypertension, tumor lysis syndrome, chronic kidney, and cardiovascular diseases. Evidence suggests that elevated uric acid levels in serum can be an independent and significant risk factor for these pathologies. Lowering uric acid levels has been shown to benefit patients with gout, chronic kidney, and cardiovascular disease. Thus, long-term monitoring and lowering the elevated serum uric acid levels may be crucial for diagnosis and effective management of chronic hyperuricemia-related diseases.

[0006] Hyperuricemia therapeutics aim to lower serum uric acid levels either by (i) competing with xanthine oxidase enzymes to reduce the production of this compound in liver (e.g., allopurinol, febuxostat), (ii) blocking uric acid transporters (e.g., URAT1, GLUT9) to prevent its reabsorption from kidneys (e.g., probenecid, benzbromarone), or (iii) metabolizing uric acid via recombinant urate oxidase activity (e.g., pegloticase, rasburicase). However, known therapies may lead to significant side effects in patients, including hypersensitivity, intolerance, abnormalities in liver function, and immune response. For example, allopurinol can cause dosedependent side effects, especially among patients with renal insufficiency. Alternative therapies for patients intolerant to allopurinol include febuxostat and benzbromarone, but these drugs can lead to abnormalities in renal function tests and even liver toxicity in some patients. For these patients, recombinant urate oxidase therapy provides an alternative. Nevertheless, these drugs proved to be suitable for short-term treatment only. Therefore, there exists a need for additional therapies to provide long-term uric acid homeostasis without these complications.

[0007] Accordingly, the present invention provides compositions and methods for monitoring of uric acid and alternative treatment of uric acid-related diseases by utilizing the intestinal tract. The compositions provided herein take advantage of the close interaction between human health and gut microbiota by modulating the composition or activity of the microbiota via genetically engineered microbes. The engineered microbes provide non-invasive, cost-effective, and on-site diagnosis and treatment for various human diseases. For instance, the present invention provides exemplary engineered human commensal bacteria as a novel method for non- invasive monitoring of uric acid levels and long-term management of hyperuricemia through the intestinal tract. As described herein, E. coli were reprogrammed to (i) monitor changes in uric acid concentration using a bioreporter module, and (ii) degrade excess uric acid found in the environment. Moreover, an in vitro model was designed with the human intestinal cell line, Caco- 2, to study the transport and degradation of uric acid in an environment mimicking the human intestinal tract. As shown herein, the compositions of genetically engineered E. coli provide longterm monitoring and degradation of uric acid and provide an essential contribution for in situ detection and elimination of hyperuricemia-causing disease biomarker through the intestinal tract.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0008] FIG. 1 shows vector DNA designs for the uric acid biosensor module. FIG. 1A depicts the nucleic acid sequence for the synthetic uric acid-responsive pucpro promoter. FIG. IB depicts the pPUCR genetic construct which is a PucR expression vector built on a pACYCl 84 backbone. FIG. 1C depicts the pucGFP genetic construct, which is a pucpro-con trolled GFP expression vector built on a pGFPuv backbone. [0009] FIGs. 2A-D show the response of engineered E. coli strains expressing green fluorescent protein (GFP) in response to changes in uric acid concentration as compared to control groups, which are (i) native E. coli without plasmids, (ii) E. coli with pPUCR vector only, and (iii) E. coli with pucGFP vector only. FIGs. 2A & B relate to a DH5a strain in M9MM media and FaSSIF-V2 media, respectively. FIGs. 2C & D relate to a K-12 MG1655 strain in M9MM media and FaSSIF-V2 media, respectively. Relative fluorescence of each sample is normalized to cell density, GD600. Each bar shows the mean normalized fluorescence and s.d. (error bars) obtained from 4 biological replicates. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates statistical significance at P < 0.001. Statistical analysis: one-way ANOVA is combined with the Bonferroni multiple comparisons test (a = 0.05).

[0010] FIG. 3A&B show characterization of uric acid bioreporter in M9MM and FaSSIF- V2 in response to increasing uric acid concentrations: 50pM, 62.5pM, 75pM, 87.5pM, lOOpM, 150pM, and 250pM. FIG. 3A relates to a DH5a strain and FIG. 3B relates to a K-12 MG1655 strain. Each data point shows the mean normalized fluorescence and s.d. (error bars) obtained from 4 biological replicates. The mean of each data point was compared to the mean of the control group, 50pM uric acid. * and *** indicates statistical significance at P < 0.05 and P < 0.001, respectively. Statistical analysis: one-way ANOVA is combined with the Bonferroni multiple comparisons test (a = 0.05).

[0011] FIGs. 4A&B show vector DNA designs for the uric acid degradation module. FIG. 4A depicts a pBR-pucLM genetic construct, which is a pucL-pucM expression vector built on pBR322 backbone. pucLM gene fusion amplified from B. subtilis 168 genome. FIG. 4B relates to a pAC-ygfU genetic construct, which is a ygfU expression vector built on pACYC184 backbone. ygfU gene amplified from E. coli K-12 MG1655 genome.

[0012] FIG. 5 shows colorimetric uric acid assay results for E. coli cells reprogrammed with uric acid degradation module components. FIGs. 5 A, 5B, and 5C relate to cells of the DH5a, K-12 MG1655, and Nissle 1917 strains, respectively. Each sample was incubated in 250pM uric acid supplemented M9MM for 24 h. Each bar shows the mean uric acid concentration and s.d. (error bars) obtained from 2 biological replicates. *** indicates statistical significance at P < 0.001 and ns indicates no statistical difference between the samples. Statistical analysis: one-way ANOVA is combined with the Bonferroni multiple comparisons test (a = 0.05).

[0013] FIGs. 6A-C show colorimetric uric acid assay results for E. coli cells reprogrammed with uric acid degradation module components. FIGs. 6A, 6B, and 6C relate to cells of the DH5a, K-12 MG1655, and Nissle 1917 strains, respectively. Each sample was incubated in 250 M uric acid supplemented FaSSIF-V2 for 24 h. Each bar shows the mean uric acid concentration and s.d. (error bars) obtained from 2 biological replicates. *** indicates statistical significance at P < 0.001 and ns indicates no statistical difference between the samples. Statistical analysis: one-way ANOVA is combined with the Bonferroni multiple comparisons test (a = 0.05).

[0014] FIG. 7A-C show uric acid transport and degradation studies using in vitro Caco- 2 model. FIG. 7A depicts an in vitro Caco-2 model for uric acid transport and degradation experiments. FIG. 7B depicts acid transport through Caco-2 monolayer over time. Elacridar supplemented samples transported significantly less uric acid from basolateral to apical side. FIG. 7C depicts apical uric acid after 2 and 6 h of incubation in the presence of bacteria. MG1655 that is engineered with both pBR-pucLM and pAC-ygfU (MG1655 pucLM & ygfU), significantly reduced apical uric acid levels. Each bar shows the mean uric acid concentration and s.d. (error bars) obtained from 6 biological replicates. * and ** indicate statistical significance at P < 0.05 and P < 0.01, respectively. Statistical analysis: unpaired student’s t-test (a = 0.05).

[0015] FIG. 8A shows characterization of uric acid bioreporter in probiotic E. coli Nissle 1917 in response to increasing uric acid concentrations: 50pM, 62.5pM, 75pM, 87.5pM, lOOpM, 150pM, and 250pM in M9MM. FIG. 8B shows comparison of bioreporter module’s performance in Nissle 1917 to K-12 MG1655. Each data point shows the mean normalized fluorescence and s.d. (error bars) obtained from 4 biological replicates. The mean of each data point was compared to the mean of the control group, 50pM uric acid. * and *** indicates statistical significance at P < 0.05 and P < 0.001 respectively. Statistical analysis: one-way ANOVA is combined with the Bonferroni multiple comparisons test (a = 0.05).

[0016] Figs. 9A-9C show FACS analysis results for BCRP expression in Caco-2, human intestinal epithelial cell line. Analyzed samples include unstained Caco-2 cells as the negative control (FIG. 9A), and replicates of Caco-2 cells that are stained with PE-conjugated anti-BCRP antibody (FIGs. 9B & C).

SUMMARY OF THE INVENTION

[0017] The present invention relates in part to an engineered host cell comprising a genetic construct comprising a reporter gene under the control of a uric acid-responsive promoter, for example a pucpro promoter. In certain embodiments, the engineered host cell expresses a uric acid-binding protein, for example a PucR protein. [0018] The present invention also relates to a method for detecting uric acid concentrations in a subject, comprising administering a composition comprising the aforementioned engineered host cell to the subject. In certain embodiments, the method comprises determining uric acid concentrations using an assay that quantifies a component of the engineered host cell.

[0019] The present invention further relates in part to an engineered host cell comprising a nucleic acid encoding a urate oxidase and/or a nucleic acid encoding a uric acid transporter.

[0020] Another aspect of the present invention is a method for decreasing uric acid concentrations in a subject in need thereof, the method comprising administering a composition comprising such an engineered host cell to the subject. In certain embodiments, the subject is in need of treatment for a uric acid-related disease, for example gout, metabolic syndrome, hypertension, tumor lysis syndrome, a chronic kidney disease, or a cardiovascular disease.

[0021] Yet another aspect of the present invention is a method for treating a subject suffering from a uric acid-release disease, the method comprising: (a) administering a composition comprising a first engineered host cell comprising a reporter gene under the control of a uric acid-responsive promoter and determining a concentration of uric acid by way of an assay quantifying a component of the recombinant host cell; and (b) administering a therapeutic amount of a composition comprising a second engineered host cell which decreases uric acid concentrations in the subject. In certain embodiments, the second engineered host cell comprises nucleic acid(s) encoding a urate oxidase and/or a uric acid transporter.

DETAILED DESCRIPTION OF THE INVENTION

[0022] This present invention relates in part to an engineered host cell which may be used as a uric acid bioreporter.

[0023] In embodiments of this invention, the host cell is engineered to comprise a genetic construct comprising a reporter gene under the control of a uric acid-responsive promoter.

[0024] In certain such embodiments, the uric acid-responsive promoter is a synthetic promoter, for example a pucpro promoter, illustrated, for example, in Figure 1 and in Supplementary Tables 2 and 3. This promoter is inspired by a similar regulatory system found in Bacillus subtilis, where genes related to purine breakdown are naturally regulated based on uric acid concentrations. The pucpro promoter was created by replacing the spacer between the -35 and -10 consensus sequences of the constitutive chloramphenicol (CAT) promoter with the pucR box from B. subtilis 168 (FIG. la). In certain embodiments, the pucpro promoter comprises the nucleic acid of SEQ ID NO: 1.

[0025] The reporter gene may, for example, encode a fluorescent reporter protein. Examples of such a protein include, but are not limited to, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, far-red fluorescent protein, and infra-red fluorescent protein. In certain embodiments, the reporter gene expresses a green fluorescent protein.

[0026] In certain embodiments, the genetic construct comprises a reporter gene encoding a green fluorescent protein under the control of a pucpro promoter. In certain such embodiments, the genetic construct is the pucGFP construct (see FIG. 1C).

[0027] In embodiments of the present invention, the engineered host cell also expresses a uric acid-binding protein.

[0028] In certain embodiments, the uric acid-binding protein is a PucR protein. The PucR protein is a transcriptional activator normally found in Bacillus subtilis and involved in the induction of the purine degradation pathway. When bound by uric acid, the PucR protein becomes activated and thus capable of binding the pucpro promoter and inducing the transcription of downstream genes.

[0029] In certain such embodiments, the engineered host cell comprises a genetic construct encoding the PucR protein, for example a pPUCR construct (see FIG. IB).

[0030] In embodiments of the present invention, the engineered host cell comprises a genetic construct comprising a reporter gene under the control of the pucpro promoter and expresses the PucR protein. In the presence of uric acid, the PucR protein is bound by uric acid and becomes activated. In activated form, it binds the pucpro promoter and inducing the transcription of the linked reporter gene. See FIG. ID.

[0031] The engineered host cell may be a bacterial cell, for example, an Escherichia coli (E. coli) cell. Examples include various strains like DH5a, K-12 MG1655, and Nissle 1917.

[0032] The present invention also relates to a composition comprising the aforementioned engineered host cell.

[0033] This present invention also provides a method for detecting uric acid concentrations in a subject. The method involves administering a composition comprising the engineered host cell of the present invention to the subject and determining uric acid concentrations using an assay that quantifies a component of the engineered host cell. [0034] In certain such embodiments, the engineered host cell expresses a fluorescent reporter protein and the assay is a fluorescence-based assay.

[0035] In some instances, the method includes monitoring a uric acid-related disease in the subject based on the determined uric acid concentration. The uric acid-related disease may be associated with hyperuricemia or hyperuricemia in the subject. In certain embodiments, the uric acid-related diseases considered is gout, metabolic syndrome, hypertension, tumor lysis syndrome, chronic kidney disease, and cardiovascular disease. In certain such embodiments, the uric acid-related disease is gout.

[0036] Another aspect of the present invention is an engineered host cell for use in lowering uric acid levels in a subject. Such a host cell may, for example, be used to lower uric acid levels in the intestines of a subject and thus prevent hyperuricemia, a condition characterized by elevated serum uric acid levels.

[0037] The engineered host cell may be a bacterial cell, for example, an Escherichia coli (E. coli) cell, which naturally occurs in the human gut microbiota. Examples include various strains like DH5a, K-12 MG1655, and Nissle 1917.

[0038] In certain embodiments, the engineered host cell comprises a nucleic acid encoding a urate oxidase, for example a B. subtilis urate oxidase. Urate oxidase plays a pivotal role in breaking down uric acid into allantoin.

[0039] In certain such embodiments, the host cell is engineered to comprise a pBR- pucLM genetic construct. This is a pucL-pucM expression vector built on pBR322 backbone (See FIG. 4A).

[0040] In certain embodiments, the engineered host cell comprises a nucleic acid encoding a uric acid transporter, for example an E. coli K-12 uric acid transporter, YgfU. In embodiments wherein the host cell is an E. coli cell, the cell may be engineered to overexpress YgfU. Uric acid transporter allows for increased uric acid uptake by the cell.

[0041] In certain embodiments, the host cell is engineered to comprise a pAC-ygfU genetic construct. This is a ygfU expression vector built on a pACYC184 backbone (See FIG. 4B).

[0042] In certain embodiments, the host cell comprises nucleic acids encoding both a urate oxidase and a uric acid transporter. In certain such embodiments, the host cell is an E. coli cell engineered to comprise the pBR-pucUM and pAC-ygfU genetic constructs. [0043] The present invention also relates to a composition comprising the aforementioned engineered host cell. The composition may be for use in decreasing uric acid concentrations in a subject in need thereof.

[0044] Another aspect of the present invention is a method for decreasing uric acid concentrations in a subject in need thereof. This method involves administering a composition comprising an engineered host cell as described above. In certain embodiments, the composition degrades uric acid in the subject, for example within the intestinal tract.

[0045] Also another aspect of the present invention is a use of a composition comprising an engineered host cell as described above in the preparation of a medicament for reducing uric acid concentrations in a subject in need thereof.

[0046] In certain embodiments of the aforementioned method and use, the subject is in need of treatment for a uric acid-related disease, for example one associated with hyperuricemia. In certain such embodiments, the uric acid-related disease is gout, metabolic syndrome, hypertension, tumor lysis syndrome, a chronic kidney disease, or a cardiovascular disease. In certain such embodiments, the uric acid-related disease is gout.

[0047] A further aspect of the present invention is a method for treating a subject suffering from a uric acid-release disease, wherein the method comprises: (a) administering a first composition comprising the engineered host cell of the present invention comprising a reporter gene under the control of a uric acid-responsive promoter and determining a concentration of uric acid by way of an assay quantifying a component of the recombinant host cell; and (b) administering a therapeutic amount of a second composition comprising the engineered host cell of the present invention to decrease uric acid concentrations in the subject. In certain embodiments, the engineered host cell of (a) also expresses a uric acid-binding protein. In certain embodiments, the engineered host cell of (b) comprises nucleic acid(s) encoding a urate oxidase and/or a uric acid transporter.

[0048] In certain embodiments of the aforementioned method, the uric acid-related disease is one associated with hyperuricemia. In certain such embodiments, the uric acid-related disease is gout, metabolic syndrome, hypertension, tumor lysis syndrome, a chronic kidney disease, or a cardiovascular disease. In certain such embodiments, the uric acid-related disease is gout.

[0049] In embodiments of the present invention, the level of uric acid may be determined using a colorimetric assay.

[0050] The following numbered embodiments are contemplated and are non-limiting: 1. An engineered host cell comprising a genetic construct comprising a reporter gene under the control of a uric acid-responsive promoter.

2. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the uric acid-responsive promoter is a pucpro promoter.

3. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the uric acid-responsive promoter comprises the nucleic acid sequence of SEQ ID NO: 1.

4. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the reporter gene encodes a fluorescent reporter protein.

5. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the reporter gene encodes a green fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a red fluorescent protein, a far-red fluorescent protein, or an infra-red fluorescent protein.

6. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the reporter gene encodes a green fluorescent protein.

7. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the genetic construct comprises a reporter gene encoding a green fluorescent protein under the control of the pucpro promoter.

8. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the genetic construct is the pucGFP construct.

9. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, expressing a uric acid-binding protein.

10. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, expressing a PucR protein.

11. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, comprising a pPUCR construct.

12. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the uric acid-responsive promoter is a pucpro promoter and the engineered host cell expresses a PucR protein.

13. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the cell is a bacterial cell. 14. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the cell an Escherichia coli cell.

15. The engineered host cell of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the cell is a DH5a, K-12 MG1655, or Nissle 1917 cell.

16. A composition comprising the engineered host cell of any one of clauses 1-15.

17. The composition of clause 16, any other suitable clause, or any combination of suitable clauses, for use in determining uric acid concentrations in a subject.

18. A method for detecting uric acid concentrations in a subject, any other suitable clause, or any combination of suitable clauses, comprising administering a composition comprising the engineered host cell of clause 16 to the subject.

19. The method of clause 18, any other suitable clause, or any combination of suitable clauses, comprising determining uric acid concentrations using an assay that quantifies a component of the engineered host cell.

20. The method of clause 19, any other suitable clause, or any combination of suitable clauses, wherein the engineered host cell expresses a fluorescent reporter protein and the assay is a fluorescence-based assay.

21. An engineered host cell, comprising a nucleic acid encoding a urate oxidase and/or a nucleic acid encoding a uric acid transporter.

22. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, wherein the host cell is a bacterial cell.

23. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, wherein the host cell is an Escherichia coli cell.

24. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, wherein the host cell is a DH5a, K-12 MG1655, or Nissle 1917 cell.

25. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, wherein the urate oxidase is a B. subtilis urate oxidase.

26. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, comprising a pBR-pucLM genetic construct.

27. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, wherein the uric acid transporter is an E. coli K-12 uric acid transporter. 28. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, wherein the uric acid transporter is YgfU.

29. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, comprising a pAC-ygfU genetic construct.

30. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, comprising a nucleic acid encoding a urate oxidase and a nucleic acid encoding a uric acid transporter.

31. The engineered host cell of clause 21, any other suitable clause, or any combination of suitable clauses, comprising a pBR-pucLM genetic construct and a pAC-ygfU genetic construct.

32. A composition comprising the engineered host cell of any one of clauses 21-31.

33. The composition of clause 32, any other suitable clause, or any combination of suitable clauses, for use in decreasing uric acid concentrations in a subject in need thereof.

34. A method for decreasing uric acid concentrations in a subject in need thereof, the method comprising administering the composition of clause 32, any other suitable clause, or any combination of suitable clauses, to the subject.

35. The method of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the subject is in need of treatment for a uric acid-related disease.

36. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the uric acid-related disease is associated with hyperuricemia.

37. The method of clause 36, any other suitable clause, or any combination of suitable clauses, wherein the uric acid-related disease is gout, metabolic syndrome, hypertension, tumor lysis syndrome, a chronic kidney disease, or a cardiovascular disease.

38. The method of clause 36, any other suitable clause, or any combination of suitable clauses, wherein the uric acid-related disease is gout.

39. A use of the composition of clause 32, any other suitable clause, or any combination of suitable clauses, for the preparation of a medicament for reducing uric acid concentrations in a subject in need thereof.

40. A method for treating a subject suffering from a uric acid-release disease, the method comprising:

(a) administering a composition comprising a first engineered host cell comprising a reporter gene under the control of a uric acid-responsive promoter and determining a concentration of uric acid by way of an assay quantifying a component of the recombinant host cell; and

(b) administering a therapeutic amount of a composition comprising a second engineered host cell which decreases uric acid concentrations in the subject.

41. The method of clause 40, any other suitable clause, or any combination of suitable clauses, wherein the second engineered host cell comprises nucleic acid(s) encoding a urate oxidase and/or a uric acid transporter.

[0051] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps

EXAMPLES

EXAMPLE 1

Materials and Methods

[0052] The instant example provides exemplary materials and methods utilized in Examples 2-5 as described herein.

Strains and growth conditions

[0053] Bacterial strains utilized in the following examples are listed in Supplementary Table 1.

Supplementary Table 1

[0054] E. coli DH5a, E. coli K-12 MG1655, and E. coli Nissle 1917 were used as hosts for plasmid construction and recombinant protein expression. For chromosomal isolation of pucL, and pucR genes, B. subtilis 168, and for the isolation of ygfU gene, E. coli K-12 MG1655 was used. All strains were maintained in Luria-Bertani (LB) broth at 37°C with 250 rpm shaking for routine growth. For GFP assays, M9 minimal medium (M9MM) and Fasted State Simulated Intestinal Fluid (FaSSIF-V2) supplemented with 0.4% glucose was prepared. For E. coli DH5a cultures during GFP assay, Ipg/ml thiamine was added into M9MM and FaSSIF-V2. For all experiments, each media type was supplemented with appropriate antibiotics: ampicillin 100 pg/ml, chloramphenicol 25 pg/ml, tetracycline 10 pg/ml, and kanamycin 25 pg/ml.

Plasmid construction

[0055] Plasmids and primers used in this study are listed in Supplementary Table 2 and 3.

Supplementary Table 2 Supplementary Table 3

* This amplification was performed in two steps: (i) pucpro-cheZ-ybaQ fusion was amplified from pSC-pucpro- cheZ template using pucproGFP-GA-Fwd and cheZ-ybaQ-GA-Rev2 primers, (ii) PCR product from step (i) was used as a template to amplify pucpro-cheZ-ybaQ for Gibson Assembly® with pSClOl backbone using pucproGFP- GA-Fwd and pSC-ybaQ-GA-Rev primer pair.

[0056] Synthetic promoter sequence, pucpro, ordered as 149 bp long, 4 nmole Ultramer® DNA Oligo from Integrated DNA Technologies (IDT). Q5® High-Fidelity DNA Polymerase (NEB®) was used inall PCR reactions. PCR amplified plasmid backbones and inserts were assembled using Gibson Assembly® Master Mix (NEB®). All plasmid sequences were verified via DNA Sanger Sequencing (Cornell Biotechnology Resource Center, Genomics Facility).

Uric acid stock

[0057] An initial stock of uric acid (50mM) was prepared by dissolving uric acid (Sigma- Aldrich®) in 400mM NaOH. If necessary, this stock solution was diluted in sterile dH2O to get desired uric acid concentrations in growth media.

GFP assay

[0058] To measure bioreporter’s performance, fluorescent assays were conducted using Synergy 4 Plate Reader, BioTek® (Winooski, VT). 485/20 excitation filter and 528/20 emission filter were used to record relative fluorescence unit (RFU) of each sample. E. coli DH5a carrying pGFPuv plasmid was used as a positive control to ensure GFP assay and plate reader settings were working fine. To induce GFP gene expression from pGFPuv, media was supplemented with 0.2mM Isopropyl P-D-l -thiogalactopyranoside JPTG) (VWR™). Single colonies of E. coli DH5a, K-12 MG1655, Nissle 1917, and engineered E. coli containing bioreporter constructs were inoculated into LB. After 18 h of incubation, cultures were diluted 1:25 in M9MM or FaSSIF-V2 supplemented with different uric acid concentrations: OpM, 50pM, 62.5pM, 75pM, 87.5pM, lOOpM, 150pM, and 250pM. Diluted cultures were grown at 37°C shaker for at least 6 h. Finally, RFU was measured for each sample and fluorescence was normalized to cell density (GD600) during data analysis.

Colorimetric uric acid assay

[0059] Single colonies of E. coli DH5a, K-12 MG1655, Nissle 1917, and engineered E. coli strains harboring uric acid degradation constructs were inoculated into LB. After 18 h of incubation, cultures were diluted 1:25 in M9MM or FaSSIF-V2 supplemented with 250pM uric acid. These diluted cultures were grown in M9MM or FaSSIF-V2 for 24 h at 37°C. The next day, overnight cultures were spun down at 3000rpm for 10 min. Supernatant was collected to measure remaining uric acid concentration in the media using colorimetric uric acid assay kit (Eton Bioscience Inc). To construct a standard curve for this colorimetric assay, a 50mM uric acid stock was prepared as described above and performed serial dilutions (OpM, 25 pM, 50pM, lOOpM, 150pM, 200pM, 250pM, and 500pM) both in M9MM and FaSSIF-V2.

Uric acid transport and degradation using in vitro Caco-2 model

[0060] Caco-2 cells were obtained from ATCC® (HTB-37™). Cells were cultured in Dulbecco’s Modified Eagle Medium - DMEM - supplemented with 10% fetal bovine serum albumin - FBS - and lx antibiotic- antimycotic solution (Gibco™) at 37°C under an atmosphere of 5% CO2 in the air. For uric acid transport and degradation studies, Caco-2 cells were seeded on 12mm, 0.4pm Transwell® inserts (Corning®). Seeded cells were cultured for 16-18 d, and Caco-2 monolayer integrity was assessed via Lucifer Yellow % passage assay before each experiment ⁴⁶ . Monolayers with < 3% LY passage were used in the following setups.

[0061] For the uric acid transport experiments, uric acid supplemented transport medium - lx Hank’s Balanced Salt Solution, lOmM HEPES pH: 7.4, 25mM glucose - was added to the basolateral compartment and transport medium without uric acid was added to the apical compartment of Transwell® inserts. For the treatment group, 25pM elacridar (Sigma- Aldrich®) - BCRP inhibitor - was dissolved in DMSO and added to the apical side. For the control group, only DMSO was added to the apical side. Monolayers were incubated at 37°C, 5% CO2 for up to 8 h, and aliquots were collected at every 2 h from apical compartment to measure the change in uric acid concentrations.

[0062] For the uric acid degradation studies on Caco-2 monolayers, 2.5mM uric acid supplemented transport medium was added to the basolateral compartment and transport medium without uric acid was added to the apical compartment. These Transwell® setups were incubated at 37°C, 5% CO2 to enable uric acid transport into the apical chamber. As for the bacteria, wildtype E. coli K-12 MG1655 and engineered strains with uric acid degradation module were grown in LB medium at 37°C for 12-16 h. Overnight cultures were spun down at 3000 rpm for 10 min, and supernatant was removed. Cell pellets was resuspended in DMEM media without phenol red (Gibco™) and with FBS (Biowest), and diluted to OD600 = 1.0. The diluted culture was added into the apical chamber of previously prepared Transwell® setups, giving the final OD600 of 0.1. These bacteria supplemented setups were incubated at 37°C, 5% CO2 for up to 6 h and aliquots (lOOpl) were collected from apical compartment to measure the change in uric acid concentrations.

FACS Analysis

[0063] Caco-2 cells were seeded on 12mm, 0.4pm Transwell® inserts (Corning®) at a density of 5 * 10 ⁴ cells / cm ² , and incubated at 37°C, 5% CO2 for 21 d. During this period, cells were cultured in 10% FBS and l x antimycotic-antibiotic supplemented DMEM, which was replaced with fresh media every 2 to 3 d. After 21 d, Caco-2 monolayer was washed with lx PBS, and cells were treated with trypsin-EDTA 0.25% solution (Gibco™) for 20 min, and centrifuged at 300 RCF for 5 min. Harvested cells were washed in ice cold FACS buffer (lx PBS, 0.5% BSA, 0.1% NaNa), and their numbers were adjusted to 1 * 10 ⁶ cells/ml. Next, Caco- 2 cells were labeled with a PE-conjugated BCRP (ABCG2) monoclonal antibody (Invitrogen, Catalog # 12-8888-42) following a FACS cell surface staining protocol.

[0064] All FACS analysis were performed using the Thermo Fisher Attune™ NxT Flow Cytometer (Cornell Biotechnology Resource Center, Flow Cytometry Facility).

EXAMPEE 2

Estimation of intestinal uric acid concentration

[0065] An estimate of uric acid concentration in the human intestinal lumen is required to develop a bioreporter, which can differentiate between healthy and disease states. Studies reported different ranges for the daily amount of uric acid removed via renal pathway. In particular, the average urinary uric acid excretion can be between 600 to 650 mg/d for healthy individuals and over 1000 mg/d for hyperuricemia patients. Using these urinary uric acid excretion values, we calculated the amount of total endogenous uric acid that the human body excretes. As 30% of that total amount is known to be excreted via intestines, we estimated the intestinal secretion rates of healthy individuals and hyperuricemia patients as 0.1786 mg/min and 0.2976 mg/min, respectively. For our calculations, we assumed that the renal to intestinal uric acid excretion ratio is the same for healthy and hyperuricemia cases. However, the intestinal uric acid secretion rate can be higher for hyperuricemia patients as intestinal secretion compensates for renal underexcretion.

[0066] To calculate steady state intestinal uric acid concentrations, we applied a mass balance on uric acid evolution in the intestine using equation (1). In this equation, mu indicates the uric acid mass in the intestine, lb/n is the intestinal volume, [U]is the uric acid concentration, s is the intestinal secretion rate, q is the intestinal flow rate, and k represents the uric acid degradation rate constant. Degradation rate constant A was set equal to three times the intestinal flow rate because within the intestines, 75% of uric acid gets degraded by resident bacteria.

[0067] Using long-term intestinal flow rate, 5 ml/min, we calculated steady state uric acid concentrations as 53.15pM for healthy individuals and > 88.57pM for hyperuricemia patients. These representative intestinal uric acid concentrations enabled us to set a threshold for healthy and disease states.

EXAMPLE 3

Engineering E. coli as uric acid bioreporter

[0068] To develop E. coli as a uric acid bioreporter, we designed a synthetic uric acid responsive promoter (pucpro) sequence. The rationale behind the design of this promoter was to adapt B. subtilis'^ purine catabolism regulation into E. coli for uric acid detection. Gram positive soil bacterium, B. subtilis, utilizes purine bases as a nitrogen source and regulates genes needed for purine catabolism via the PucR protein. Two factors are important for PucR mediated transcriptional regulation: (i) the position of the PucR binding site on the genome relative to transcription start point, and (ii) the presence of purine degradation products, such as uric acid, acting as an effector molecule for PucR activation ³⁴ . To build the pucpro promoter, we replaced the spacer between -35 and -10 consensus sequences of constitutive chloramphenicol (CAT) promoter with pucR box from B. subtilis 168 (FIG. 1A). Our bioreporter module comprises two constructs, (i) pPUCR and (ii) pucGFP, which enable PucR protein production and uric acid induced green fluorescent protein (GFP) expression, respectively (FIGs. IB, C).

[0069] Laboratory strain E. coli DH5a and human commensal strain E. coli K-12 MG1655 were used as hosts for our uric acid bioreporter module. To quantify the bioreporter’s performance in these bacteria, relative GFP fluorescence of each sample was measured and normalized to cell density. In the first GFP assay (FIG. 2), the bioreporter and control strains response to changes in uric acid concentration (OpM, 50pM, and 250pM) was compared under two conditions: (i) M9 minimal media (M9MM) to ensure that uric acid is the effector molecule, and (ii) fasted state simulated intestinal fluid (FaSSIF-V2) to mimic human intestinal fluid conditions in vitro. Both DH5a (FIGs. 2A, B) and K-12 MG1655 (FIG. 2C, D) bioreporter strains produced significantly more GFP protein (P<0.001 ) when uric acid in the environment increased from 50pM to 250pM. The control group carrying the pucGFP vector had the same type of increase in GFP production (FIG. 2B-D), but this response was significantly less (P<0.001) compared to combined effect of pPUCR and pucGFP in the cell. On the other hand, native E. coli and E. coli carrying the pPUCR vector, did not show any difference in their GFP fluorescence in response to changing uric acid levels. Results indicate (FIG. 2) that PucR protein induces transcription from the pucpro promoter in the presence of uric acid. Without being bound by any theory, this type of positive regulation can demonstrate conformational changes on DNA, enhancing RNA polymerase machinery’s assembly following PucR binding to pucpro.

[0070] In the second round of GFP assays (FIG. 3), the bioreporter module was characterized in M9MM and FaSSIF-V2 media for its sensitivity to increasing uric acid concentrations (50pM, 62.5pM, 75pM, 87.5pM, lOOpM, 150pM, and 250pM). Linear regression analysis enabled us to calculate the goodness of fit (R ² ), as well as study the relationship between normalized GFP fluorescence and increasing uric acid concentrations. Results showed that the bioreporter module in both bacterial strains can detect changes in uric acid in a dose-dependent manner. Engineered DH5a significantly differentiated (P<0.001 ) between healthy (~53pM) and disease state (> 88pM) intestinal threshold values both in M9MM and FaSSIF-V2 (FIG. 3A). Engineered K-12 MG1655 significantly differentiated (P<0.05) between healthy and disease states in M9MM (FIG. 3B). However, compared to DH5a, bioreporter module in K-12 MG1655 was less sensitive to changes in uric acid under simulated intestinal fluid (FaSSIF-V2) conditions. In this media, MG1655 only managed to differentiate (P<0.001) between low (50pM) and high levels of uric acid (>150pM) (FIG. 3B).

[0071] Using the same experimental setup and cloning as for MG1655 and DH5a, we tested the probiotic E. coli Nissle 1917, as well; results suggested that Nissle may not provide robust findings as a uric acid bioreporter.

EXAMPLE 4

Engineering E. coli to lower serum uric acid levels

[0072] Clinical trials with a uric acid absorbing hydrogel and recent studies in animal models demonstrated the important role of intestinal uric acid clearance in hyperuricemia prevention. To regulate serum uric acid levels via the intestinal lumen, we reprogrammed E. coli, components of gut microbiota, for enhanced uric acid degradation. Our uric acid degradation module comprises two constructs: (i) pBR-pucLM (FIG. 4A) enables constitutive expression of recombinant B. subtilis urate oxidase in E. coli for degradation of uric acid into allantoin, and ( ii ) pAC-ygfU (FIG. 4B) enables overexpression of E. coli K-12 uric acid transporter, YgfU, for increased uptake of uric acid into the cell.

[0073] E. coli strains DH5a, MG1655, and the probiotic Nissle 1917 were used as host strains for our uric acid degradation module. These engineered bacteria along with the control groups were tested in 250pM uric acid supplemented M9MM and FaSSIF-V2 media. To measure the amount of uric acid remaining in M9MM and FaSSIF-V2 after 24 h of incubation, colorimetric uric acid assays were performed on each sample. As a negative control group, uric acid supplemented media with no cells enabled us to ensure that uric acid was stable in these growth conditions until the colorimetric assay was performed. Assay results demonstrated that reprogrammed E. coli DH5a (FIGs. 5A, 6A), E. coli K-12 MG1655 (FIGs. 5B, 6B), and E. coli Nissle 1917 (FIGs. 5C, 6C) degraded all the uric acid found in the environment after 24 h of incubation in M9MM and FaSSIF-V2, which was significantly lower (P<0.001 ) compared to wild-type E. coli strains and negative control groups harboring pBR-pucLM or pAC-ygfU construct only.

EXAMPLE 5

Designing an in vitro Caco-2 model for uric acid transport and degradation

[0074] Efflux transporter, BCRP, is expressed abundantly at the apical membrane of human small intestinal epithelial cells, and it contributes to intestinal excretion of uric acid. Studies on animal models demonstrated that lack of BCRP causes decrease in intestinal uric acid secretion, and consequently a significant increase in serum uric acid levels. Based on BCRP’s crucial role in intestinal uric acid transport, we designed an in vitro model of human intestinal epithelium using the BCRP expressing cell-line, Caco-2. This human intestinal epithelial cellline shows polarized uric acid flux from basolateral to apical side, mimicking the excretion of uric acid into human intestinal lumen.

[0075] Using this model (FIG. 7A), we studied uric acid transport and its degradation via our engineered E. coli strain K-12 MG1655 in an environment that mimics the human intestinal tract.

[0076] To conduct the uric acid transport and degradation experiments on Transwell® inserts, we first analyzed the BCRP expression on our Caco-2 cultures via FACS. These results verified the abundant expression levels of BCRP on this human intestinal epithelial cell line. Next, we examined the uric acid transport across the Caco-2 monolayer. We supplemented the apical side with potent BCRP inhibitor, elacridar, to demonstrate that uric acid transport across the monolayer is predominantly due to BCRP activity. Colorimetric uric acid assay results (FIG. 7B) demonstrated that incubating the monolayer and supplementing the basolateral compartment with 2.5mM uric acid achieved uric acid levels greater than46.44pM on the apical side. Moreover, after 2~6 h incubation with elacridar-supplemented Caco-2 monolayers transported 46.7-56.2% less (P<0.01) uric acid from the basolateral to apical side (FIG. 7B). This demonstrated the importance of BCRP-mediated uric acid transport for polarized flux of this molecule.

[0077] In the second experimental setup, we investigated the uric acid degradation potential of engineered E. coli strain K-12 MG1655, which was carrying pBR-pucLM and pAC- ygfU constructs. Based on the data from uric acid transport experiments, we incubated the Caco- 2 monolayer with 2.5mM uric acid and bacteria for 6 h, uric acid concentrations were measured via colorimetric uric acid assay. Results demonstrated that apical chambers with engineered E. coli K-12 MG1655 (MG1655 pucLM & ygfU) had 40.35% less (P<0.01) uric acid compared to wild-type MG1655 after 6 h. (FIG. 7C).