Inventors: Thomas T. Eng, Deepanwita Banerjee, Aindrila Mukhopadhyay

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 62/980,054, filed February 21, 2020, which is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT [0002] The invention was made with government support under Contract Nos. DE-AC02- 05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention is in the field of the production of indigoidine.

REFERENCE TO A "SEQUENCE LISTING" SUBMITTED AS ASCII TEXT FILES

VIA EFS-WEB

[0004] The Sequence Listing written in file 2019-145-03_Sequence_Listing_ST25.txt created on Feb. 22, 2021, 12,697 bytes, machine format IBM-PC, MS-Windows operating system, in accordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0005] Heterologous production of bioproducts has been demonstrated for a very large number of compounds and in a wide variety of microbial hosts ^1,2 . Yet, even the most well- designed heterologous pathway requires considerable additional work to reach production titers rates and yields (TRY) necessary for the adoption of these systems in the industry ^3,4 . In addition, the production parameters of a strain at lab-scale is often not predictive of its performance and robustness when cultivated in different modes or higher scales, and several approaches from a practical or process engineering standpoint have been described ^5,6 . These issues could be attributed to the intrinsic competition for carbon between the native metabolism and the heterologous pathway, leading to phenotypic drift in diverse growth modes. As a result, only a small fraction of bioproduction strains have been successfully scaled and deployed ² .

[0006] Indigoidine is a natural blue pigment natively produced by several bacteria via biosynthetic gene clusters. This 3’,3’-bipyridyl pigment is formed through condensation of two molecules of L-glutamine catalyzed by a non-ribosomal peptide synthetase (NRPS). NRPS are large assembly-line enzymes and are organized in modules that are each responsible for the introduction of one L-amino acid to the NRP. Each module consists of several domains with defined functions that synthesize NRPs in a sequential multi-step process. The secondary metabolite class of NRPs includes molecules with a range of pharmaceutical applications, such as immunosuppressants, antibiotics, anticancer drugs and antiviral compounds. However, the low NRP production levels from native hosts and their complex chemical structures impede mass production by purification from biological material or chemical synthesis. Furthermore, despite the availability of biosynthetic tools for metabolic engineering and pathway discovery, optimization of NRP production in their natural hosts remains challenging.

SUMMARY OF THE INVENTION

[0007] The present invention provides for a genetically modified bacterial host cell capable of producing indigoidine, wherein the host cell comprises a non-ribosomal peptide synthetase (NRPS) that converts glutamine to indigoidine, and the bacterial host cell is reduced in its expression of one or more of the following enzymes or enzymes catalyzing the indicated reactions: Glucose dehydrogenase (ubiquinone 8 as acceptor, periplasm); Phosphoenolpyruvate synthase; Malate dehydrogenase; Malate dehydrogenase (ubiquinone 8 as acceptor); Malic enzyme NADP; Transaldolase; N acetylornithine deacetylase; Ornithine Decarboxylase; Proline dehydrogenase; Poly 3 hydroxyalkanoate polymerase 3 Hydroxybutanoyl CoA; 1,6 anhydrous N Acetylmuramate kinase; D lactate transport via proton symport periplasm; Carboxylic acid dissociation; and HC03 equilibration reaction, , and any other described in Table 4, and any other genes described in Supplemental Tables 5 and 6 of U.S. Provisional Patent Application Ser. No. 62/980,054, filed February 21, 2020, which is incorporated by reference in its entirety.

[0008] In some embodiments, the bacterial host cell is a proteobacteria cell. In some embodiments, the proteobacteria cell is a Gammaproteobacteria cell. In some embodiments, the Gammaproteobacteria cell is a Pseudomonadales or Enterobacterales cell. In some embodiments, the Gammaproteobacteria cell is a Pseudomonadales cell, which is a Pseudomonadaceae cell. In some embodiments, the Pseudomonadaceae cell is a Pseudomonas, Azotobacter , Mesophilobacter , Oblitimonas , Permianibacter , Rugamonas, or Thiopseudomonas cell. In some embodiments, the Pseudomonas cell is a P. putida , P. aeruginosa , I chlororaphis , I fluorescens , I pertucinogena , I stutzeri , I syringae, P. cremoricolorata, P. entomophila , I fulva , I monteilii , I mosselii , I oryzihabitans, P. parafluva , or P. plecoglossicida. In some embodiments, the Gammaproteobacteria cell is a Enterobacterales cell, which is a Enterobacteriaceae cell. In some embodiments, the Enterobacteriaceae cell is an Escherichia , Enter obacillus, Enterobacter , Klebsiella , Salmonella , or Shigella cell. In some embodiments, the Escherichia cell is an E. coli , E. albertii , E. fergusonii , E. hermanii , E. marmotae, or E. vulneris. In some embodiments, the host cell is a Gram negative bacterium. In some embodiments, the host cell is a bacterium from the Azotobacter , Escherichia, Salmonella, Vibrio, Pasteurella , Haemophilus , or Pseudomonas genus. In some embodiments, the host cell is a bacterium from the species Escherichia coli, Salmonella enterica, Vibrio cholerae, Pasteurella multocida , Haemophilus influenza , Pseudomonas putida , or Pseudomonas aeruginosa.

[0009] In some embodiments, the bacterial host cell in its unmodified state in nature comprises one or more genes encoding the following enzymes or enzymes catalyzing the indicated reactions: Glucose dehydrogenase (ubiquinone 8 as acceptor, periplasm); Phosphoenolpyruvate synthase; Malate dehydrogenase; Malate dehydrogenase (ubiquinone 8 as acceptor); Malic enzyme NADP; Transaldolase; N acetylornithine deacetylase; Ornithine Decarboxylase; Proline dehydrogenase; Poly 3 hydroxyalkanoate polymerase 3 Hydroxybutanoyl CoA; 1,6 anhydrous N Acetylmuramate kinase; D lactate transport via proton symport periplasm; Carboxylic acid dissociation; and HC03 equilibration reaction. In some embodiments, the bacterial host cell in its unmodified state in nature comprises two or more, three or more, four or more, five or more, six of more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, or all of the genes described herein.

[0010] In some embodiments, the NRPS is heterologous to the host cell. In some embodiments, the NRPS is a bacterial NRPS. In some embodiments, the NRPS is a Streptomyces lavendulae NRPS (BpsA). [0011] In some embodiments, the genetically modified bacterial host cell is reduced in its expression of two or more, three or more, four or more, five or more, six of more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, or all of the enzymes or enzymes catalyzing the indicated reaction indicated herein.

[0012] In some embodiments, the reduced in its expression of one or more of the following enzymes or enzymes catalyzing the indicated reactions includes, but is not limited to, reduced transcription of a gene by any suitable means (such as gene silencing, antisense, replacement of a native promoter with a promoter with a lower transcription rate, introduction of one or more repressor sites, replacement or elimination of an activation site, or the like), reduced translation of the transcript of a gene by any suitable means (such as an altered ribosomal binding site that has a reduced ability to bind to a ribosome), or the deletion of a part of all of the native promoter, or the deletion of a part of all of the open reading frame of the gene, or any means taught herein. Gene silencing includes RNAi, CRISPR, or siRNA. The reduction of transcription, translation, and/or gene expression can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any range of value between any two preceding values, including the stated values.

[0013] The present invention provides for a method for a genetically modified bacterial host cell producing indigoidine, comprising (a) providing a genetically modified bacterial host cell of the present invention, (b) culturing or growing the host cell in a suitable culture or medium such that indigoidine is produced, and (c) optionally extracting or separating the indigoidine from the rest of the culture or medium, and/or host cell.

[0014] In some embodiments, the providing step (a) comprises introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell. In some embodiments, the culturing or growing step (b) comprises the host cell growing by respiratory cell growth. In some embodiments, the culturing or growing step (b) takes place in a batch process or a fed-batch process, such as a high-gravity fed-batch process. In some embodiments, the culture or medium comprises hydrolysates derived or obtained from a biomass, such as a lignocellulosic biomass. In some embodiments, the culture or medium comprises one or more carbon sources, such as a sugar, such as glucose or galactose, or glycerol, or a mixture thereof. In some embodiments, the carbon source is fermentable. In some embodiments, the carbon source is non-fermentable. In some embodiments, the culture or medium comprises urea as a nitrogen course.

[0015] The present invention provides for a method for constructing a genetically modified bacterial host cell of the present invention, comprising (a) introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell.

[0016] In some embodiments, the NRPS is heterologous to the host cell. In some embodiments, the NRPS is a bacterial NRPS. In some embodiments, the NRPS is a Streptomyces lavendulae NRPS (BpsA). In some embodiments, the NRPS comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein the NRPS comprises the enzymatic activity to convert glutamine to indigoidine.

[0017] The present invention provides for a method for a genetically modified bacterial host cell producing indigoidine, comprising (a) providing a genetically modified bacterial host cell of the present invention, (b) culturing or growing the host cell in a suitable culture or medium such that indigoidine is produced, and (c) optionally extracting or separating the indigoidine from the rest of the culture or medium, and/or host cell.

[0018] The present invention provides for a method for constructing a genetically modified bacterial host cell of the present invention, comprising (a) introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell.

[0019] Indigoidine is a redox active blue pigment that has documented use as a dye. It can also serve as a respiration signal in cultivation optimizations and has the chemical structure of a molecule that can be used in development of biomaterials (e.g. polymers). The pigment may be used to report the redox and respiratory state of a large culture that may be critical for production performance. In some embodiments, the invention comprises the use of a heterologous codon-optimized version of an NRPS in a bacterial host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0021] Figure 1 A. Product Obligatory Strain Engineering (PrOSE) workflow diagram. PrOSE can potentially be extended to any carbon source, host and/or metabolite. Inputs specific to this specific host/fmal product PrOSE work is marked in green font.

[0022] Figure IB: The central metabolism of Pseudomonas putida is engineered to produce indigoidine from either glucose or galactose. Heterologous genes are indicated in purple text. Indigoidine is derived from the TCA intermediate alpha-ketoglutarate (AKG) via two molecules of glutamine. The necessary metabolic reactions targeted for gene knockdown by dCpfl/CRISPRi are indicated with red X marks. Additional gene targets outside of P. putida central metabolism are indicated in the box on the bottom right. A total of 14 genes are targeted for CRISPR interference.

[0023] Figure 2A: Characterization of the PrOSE strain via RNAseq and Proteomics. P. putida harboring a genomically integrated indigoidine expression cassette and either an empty vector (control strain) or a dCpfl/CRISPRi targeting array examined for gene knockdown efficiency. Figure 2A. RNAseq analysis of plasmid-borne gRNA array in P. putida.

[0024] Figure 2B. Knockdown efficiency of a representative gene locus targeted for inhibition over a 72 hour time course. RNA expression levels (right hand panel) are validated with high resolution targeted proteomic analysis (left hand panel).

[0025] Figure 2C. dCpfl/CRISPR interference causes global RNA expression level changes. Volcano plot of mRNA expression levels compared at t=0h and t=24h between PrOSE and control strains.

[0026] Figure 2D. Validation of PrOSE substrate coupled predictions. PrOSE predicts that glucose/indigoidine rewiring blocks growth of engineered strains on lysine as a carbon source.

[0027] Figure 3 A. PrOSE Can Improve Titer Rate and Yield (TRY) Across Two Carbon Sources. Analysis of P. putida galETKM PrOSE strains and a control strain ( . putida galETKM , empty vector plasmid) for production of indigoidine using galactose as the sole carbon source in minimal salt media. The culture format assessed is indicated above each panel.

[0028] Figure 3B. Analysis of P. putida PrOSE and a control strain for indigoidine production grown on M9 glucose. A fed-batch feeding regime is implemented in the ambr250 cultivation format. Glucose feeding is indicated by the gray shaded area. Control samples indicated with black outlined bars or black dots. The PrOSE strains are indicated with blue bars or blue dots.

[0029] Figure 3C. Analysis of indigoidine yield across cultivation formats for both glucose fed and galactose fed strains. Yield from the control strain is indicated with black bars, and the PrOSE strain is indicated with green bars.

[0030] Figure 4A. Optimizing carbon/nitrogen ratio yield only modest improvements to indigoidine production for glucose.

[0031] Figure 4B. Optimizing carbon/nitrogen ratio yield only modest improvements to indigoidine production for (NH ₄ ) ₂ SO ₂

_[ 0032] Figure 4C. Optimizing carbon/nitrogen ratio yield only modest improvements to indigoidine production for glucose and (NH ₄ ) ₂ SO _{2 .}

[0033] Figure 4D. The basal production of Indigoidine in P. putida is 2.3 g/L indigoidine from 10 g/L glucose after 24 hours.

[0034] Figure 4E. Minimal 100 bp promoter sequences from native tRNA ligases are sufficient to express mCherry fluorescent protein, confirming that heterologous mRNA transcripts for gRNAs would be generated under M9 glucose media.

[0035] Figure 5. Standard curve used for indigoidine quantification. R squared is 0.9843. Equation: Y = 0.004723*X + 0.01645. Working equation: Y = 211.729833X - 3.48295575. Y(mg/L) Indigoidine = 211.729833 A ₆₁₂ - 3.48295575. y(g/L) Indigoidine = 0.212 A ₆₁₂ - 0.0035.

[0036] Figure 6. NMR Spectra.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0038] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0039] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0040] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like.

[0041] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0042] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0043] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0044] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like.

[0045] The term “about” refers to a value including 10% more than the stated value and 10% less than the stated value.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0047] The terms “host cell” is used herein to refer to a living biological cell that can be transformed via insertion of an expression vector.

[0048] The term "heterologous" as used herein refers to a material, or nucleotide or amino acid sequence, that is found in or is linked to another material, or nucleotide or amino acid sequence, wherein the materials, or nucleotide or amino acid sequences, are foreign to each other (i.e., not found or linked together in nature).

[0049] The terms "expression vector" or "vector" refer to a compound and/or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Particular expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence.

Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.

[0050] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g ., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. "Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

[0051] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans- acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis- acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start codon (i.e., including the 5' untranslated region of the mRNA, typically comprising 100-200 bp). Most often the core promoter sequences lie within 1-2 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. A promoter used in an expression construct of the invention is referred to by the name of the gene. Reference to a promoter by name includes a wildtype, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species.

[0052] A polynucleotide is "heterologous" to a host cell or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter ( e.g ., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).

[0053] The term "operatively linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0054] The present invention includes the following: using Pseudomonas putida KT2440 and a minimal cut set modeling to predict genes that when deleted would to lock growth to indigoidine (or glutamine) production. Very strongest conditions are used to strongly lock the growth with production. The resulting model predicted 16 genes would redirect metabolic flux to favor the desired precursor, indigoidine. The genes that needed to be edited included both non-essential and essential genes. Since essential genes cannot be deleted, an inducible, endonuclease-dead CRISPR based strategy is used to simultaneously target genes for repression. This present invention includes the resulting engineered strain and the sequential approach taken to maximize the production to theoretical max, fast rates and also low variation. This present invention includes the following embodiments:

1. Use of a genomically integrated bioproduction pathway in P. putida KT2440.

2. Optimization of cultivation conditions to maximize production.

3. Use of a metabolic modeling approach to predict a set of genes that were hitherto never implicated in the production and the use of an inducible, endonuclease-dead (dCpfl) CRISPR approach for the first time to edit all 16 genes, or any other set of genes, predicted.

[0055] In a particular embodiment, the genetically modified bacterial host cell is capable of the production of indigoidine using 1% (56mM) glucose of up to about 7.2 g/L, which is about 98% of the theoretical max yield from glucose (n=6), from reaction stoichiometry and redox balance (at 48 hours, CV = 6.9%, n=6). In a 2L fed-batch production campaign, the growth coupled strain produces about 44.4 g/L indigoidine from 60 g/L glucose, which is 74% theoretical max yield from this carbon source.

[0056] This approach has allows for a novel systematic approach to reach near the theoretical yield using a set of predicted steps. This is a highest known yield of this compound from glucose as a sole carbon source.

[0057] In some embodiments, bacterial host cell is P. putida KT2440 (Biorxiv DOI number: 10.1101/139121), which is publicly available.

[0058] The basic concept of constrained minimal cut set (cMCS) is published and can be found in the webpage for “doi.org/10.1016/j.ymben.2010.12.004”. Software code for cMCS analysis, all calculations are done using API functions of CellNetAnalyzer (webpage for: doi.org/10.1186/1752-0509-1-2) on MATLAB 2017b platform using CPLEX 12.8 as the MILP solver. An example script to calculate cMCS for growth-coupled product synthesis available at: the webpage for: mpi-magde- burg.mpg.de/projects/cna/etcdownloads.html, which is modified for P. putida KT2440 and substrates other than glucose (xylose, p- coumarate and galactose).

[0059] During strain engineering for bioproduction, maximizing the metabolic output remains unsystematic. Robust production often relies on good growth though production and growth are often at odds with each other, leading to phenotypic drift and eventually unstable/ unreliable production. To disrupt this paradigm, an approach is developed to engineer a bacterial production strain where growth and the production of the desired final product are strongly locked. Specifically, in Pseudomonas putida KT2440 a minimal cut set modeling is used to predict genes that when deleted would to lock growth to indigoidine (or glutamate) production. Very strongest conditions are used to strongly lock the growth with production. The resulting model predicts 16 genes would redirect metabolic flux to favor the desired precursor, indigoidine. The genes that needed to be edited included both non-essential and essential genes. Since essential genes cannot be deleted, an inducible, endonuclease-dead CRISPR based strategy is used to simultaneously target genes for repression.

[0060] By taking a stringent modeling approach one predicts gene edits that would strongly lock growth with production. In other words, if there is no production there will be no growth (rather than the reverse). This forces metabolic flux to be maintained towards the pathway of choice. In order to develop the resulting strain - reduction to practice - that could demonstrate this phenotype, first one has to generate a baseline strain with low variability and cultivation/ production parameters. In some embodiments, the method comprises one or more of the following:

[0061] (1) Integrating the gene pathway leads to more reproducible final titers of indigoidine compared to a plasmid-bome pathway. In some embodiments, expression of the heterologous pathway in P. putida using pTE302 (integrated indigoidine production) vs. pTE252: (plasmid-bome). The genomically-integrated heterologous gene pathway results in <10% variation (CV of this sample is 8.98%, n=12). With a plasmid borne system, coefficient of variation is 57.5%, n=12

[0062] (2) The optimal starting concentration of glucose for the production of indigoidine is 1% (56mM) glucose and between 30 mM-100 mM (NEL^SCE in M9 minimal salt media. This is based on optimization of media conditions that leverages our expert knowledge and relates to a relationship between starting glucose concentration and indigoidine titer.

[0063] (3) A growth locked strategy for the production of indigoidine results in metabolic flux to reach 98% of the calculated maximum theoretical yield. Per our minimal cut set modeling, the Growth Locked Strategy to Maximize Intracellular Glutamine for the Production of Indigoidine identify 16 genes to be deleted. These 16 genes are involved in 14 reactions that belonged to the only minimal cut set predicted for a minimum product yield of 80% and minimum biomass yield of 10%. The indigoidine biosynthesis reactions are added to the P. putida KT2440 model and checked for growth locked production of indigoidine. These genes targets have never been predicted for such an application before and are a novel claim. These genes include essential genes as are: PP 1444, PP 2082, PP 0654, PP 1251, PP 5085, PP 2168, PP 5186, PP 0864, PP 4947, PP 5003, PP 0434, PP 4735, PP 2925, PP 5005, PP 0100, and PP 0751.

[0064] To address editing a large number of genes including essential genes a dCpfl endonuclease variant was developed for a dCpfl/CRISPRi based gene targeting approach in P. putida KT2440. pTE314 is a dCpfl plasmid for growth coupled strategy to maximize glutamine production. pTE327 is a revised dCpfl plasmid, growth coupled for glutamine, no essential genes targeted. Using a multiplex CRISPR interference strategy all 16 genes are targeted for gene silencing.

[0065] Result: As predicted by the model - the engineered strain with 16 deleted genes and a genomically integrated production pathway had massive improvement of indigoidine production rate, yield, titer, and reproducibility). The production of indigoidine using 1% (56mM) glucose was 7.2g/L, which is 98% of the theoretical max yield from glucose (n=6), from reaction stoichiometry and redox balance (at 48 hours, CV = 6.9%, n=6). In a 2L fed- batch production campaign, the growth coupled strain produced 44.4g/L indigoidine from 60g/L glucose, which is 74% theoretical max yield from this carbon source. This is the first reduction to practice of a theoretical growth coupled strategy targeting more than 3 genes for repression. Directly, this provides a highly reliable and high efficiency platform for the production of Indigoidine in a bacterial host P. putida from glucose. Indigoidine has direct uses in dye and pigment industry.

[0066] The approach for the development of this host provides a general route for increasing yield and reducing variation in heterologous product formation in P. putida and other hosts.

[0067] Directly, for the production of Indigoidine it provides a platform that uses renewable carbon sources and reaches close to theoretical max. It is also a highly stable system due to its growth being genetically coupled to production of Indigoidine. Unlike most other systems - in this engineered strain the exact levels of glucose to be used and the amount of product formed can be predicted and will result superior process downstream product development.

[0068] This approach for host development provides a rationale for synchronizing the host metabolism with the heterologous pathway. It results in a superior production system for increasing yield and reduces variation in heterologous product formation. Predicting the timeframe and productivity of such a host is greater than hosts that have not been synced in this manner. Engineering the host in this manner may also overcome natural drifts that occur due to mutations, because in this strain production is required for growth.

[0069] As alternate CRISPR systems are characterized, multiple CRISPR proteins can be expressed in the same cell, each under the control of a different carbon-responsive promoter. Strong growth coupling strategies can be implemented for two different carbon sources, such as glucose and xylose. The expression of a xylose specific CRISPR (i.e., Cas9) would also inactivate the other CRISPR system (in this case, Cpfl), ensuring that only one growth coupling strategy is used even in the presence of multiple carbon sources.

[0070] The present invention provides for a genetically modified bacterial host cell capable of producing indigoidine, wherein the host cell comprises a non-ribosomal peptide synthetase (NRPS) that converts glutamine to indigoidine. [0071] In some embodiments, the NRPS is heterologous to the host cell. In some embodiments, the NRPS is a bacterial NRPS. In some embodiments, the NRPS is a Streptomyces lavendulae NRPS (BpsA). In some embodiments, the NRPS comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein the NRPS comprises the enzymatic activity to convert glutamine to indigoidine.

[0072] In some embodiments, the NRPS comprises a conserved domain, such as the amino acid sequence APRTETEKEI AEVWAKSLRR ESVSVQDDFF ESGGNSLIAV GLIRELNSRL GVSLPLQSVL ESPTVEKLSR RLEREV (SEQ ID NO:2), at the position corresponding to 937 to 1012 of SEQ ID NO : 1.

[0073] In some embodiments, the NRPS comprises a coiled coil structure, such as the amino acid sequence SRRLEREV (SEQ ID NO: 3), or SRRLEREVAQES SRLVRLHAE (SEQ ID NO:4), at the position corresponding to 1005 to 1012, or 1005 to 1025, of SEQ ID NO:l, respectively.

[0074] In some embodiments, the serine at position 972 is a modified serine, such as O- (pantetheine 4'-phosphoryi)serine.

[0075] The amino acid sequence of Streptomyces lavendulae BpsA is as follows:

10 20 30 40 50

MTLQETSVLE PTLRGTTTLP DLLAKRVAEH PEATAVAYRD EKLTYRELAS

60 70 80 90 100

RSSALAEYLR HLGVSTDDCV GLFVEPSIDL MVGAWGILSA GAAYLPLSPE

110 120 130 140 150

YPEDRLRYMI ENSQAKIILA QQRLVTRLRE LAPQDVRVVT LRESEAFVLP 160 170 180 190 200

EGQVAPAIEG ARPDSLAYVI YTSGSTGKPK GVMIEHHSIV SQLGWLRETY

210 220 230 240 250

GIDRSKTILQ KTPMSFDAAQ WEILSPANGA TW MGAPGVY ADPEGLIETI

260 270 280 290 300

VKYGVTTLQC VPTLLQGLLD TEKFPECTSL QQIFSGGEAL SRLLAIQTTQ

310 320 330 340 350

EMPGRALINV YGPTECTINS SSYAVDPAEL GEAPQSISIG APVADTEYHI

[0076] In some embodiments, the host cell comprises a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell. In some embodiments, the encoding of the NRPS to the nucleic acid is codon optimized to the bacterial host cell. In some embodiments, the nucleic acid is vector or replicon that can stably reside in the host cell. In some embodiments, the nucleic acid is stably integrated into one or more chromosomes of the host cell.

[0077] The present invention provides for a method for a genetically modified bacterial host cell producing indigoidine, comprising (a) providing a genetically modified bacterial host cell of the present invention, (b) culturing or growing the host cell in a suitable culture or medium such that indigoidine is produced, and (c) optionally extracting or separating the indigoidine from the rest of the culture or medium, and/or host cell.

[0078] In some embodiments, the providing step (a) comprises introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell.

[0079] In some embodiments, the culturing or growing step (b) comprises the host cell growing by respiratory cell growth. In some embodiments, the culturing or growing step (b) takes place in a batch process or a fed-batch process, such as a high-gravity fed-batch process. In some embodiments, the culture or medium comprises hydrolysates derived or obtained from a biomass, such as a lignocellulosic biomass. In some embodiments, the culture or medium comprises one or more carbon sources, such as a sugar, such as glucose or galactose, or glycerol, or a mixture thereof. In some embodiments, the carbon source is fermentable. In some embodiments, the carbon source is non-fermentable.

[0080] The present invention provides for a method for constructing a genetically modified bacterial host cell of the present invention, comprising (a) introducing a nucleic acid encoding the NRPS operatively linked to a promoter capable of expressing the NRPS in the host cell into the host cell. [0081] One can modify the expression of a gene encoding any of the enzymes taught herein by a variety of methods in accordance with the methods of the invention. Those skilled in the art would recognize that increasing gene copy number, ribosome binding site strength, promoter strength, and various transcriptional regulators can be employed to alter an enzyme expression level.

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[0083] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0084] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0085] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.

EXAMPLE 1

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of nonnative products at scale

[0086] Achieving high titer rates and yields (TRY) remains a bottleneck for the production of heterologous products in microbial systems, requiring elaborate engineering and many iterations. Reliably scaling engineered strains also remains risky and is rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging due to the inherent trade-off between cellular use of carbon towards growth and target metabolite production. It is hypothesized that being able to strongly couple product formation with growth may enable both high TRY and reliability across scale. In this Example, elementary mode analysis is used to predict metabolic reactions that could be targeted to lock the production of indigoidine, a sustainable pigment, with the growth of the chosen host, P. putida KT2440. The set of 16 predicted reactions is then filtered using -omics data and the final 14 gene knockdowns is implemented using a CRISPRi method optimized for/ ¹ putida. It is demonstrated that product obligatory regime could be implemented, and the 14-gene engineered P. putida strain could achieve high TRY. Further, due to dependence on product formation, the strain maintains its high TRY phenotype across scale. This approach is called PrOSE: product obligatory strain engineering. The P. putida PrOSE strain, in one design cycle, is able to demonstrate close to 50% maximum theoretical yield (0.33 g/g of glucose consumed), reaching 25.6 g/L indigoidine from glucose and shifted production from stationary to exponential phase. These desirable phenotypes are maintained from batch to fed batch, and from lab to 250ml AMBR to 2L bioreactor.

[0087] In this Example, it is explored if it is possible to rewire the metabolism of the host strain such that production of a final product or a key intermediate becomes obligatory for its growth, thereby maximizing and maintaining productivity at scale.

[0088] Native microbial processes that take such an obligatory production route include production of ethanol and organic acids during fermentation. Production of these metabolites are required during fermentative growth, and correspondingly these compounds represent the most prominent examples of successful high-volume bioproduction ^7,8 . It is hypothesized that obligatory production to growth is implementable for a heterologous product, and that such coupling could provide the desirable production characteristics. These key characteristics are the ability to reach high TRY and the ability to maintain production parameters across different growth modes and scales. A general approach for success in these two aspects represents a new paradigm for strain engineering that can directly achieve a production system with genuine industrial relevance.

[0089] The availability of comprehensive metabolic models and genome editing tools in a wide variety of microbes suitable for industrial use provides the foundation for PrOSE. The production of indigoidine, a bipyridyl compound derived from glutamine, is used as the target heterologous product. Both as a sustainable replacement for blue pigments ⁹ in a wide array of applications as well as a model non-ribosomal peptide ¹⁰ , this compound provides a valuable target to explore. Pseudomonas putida KT2440 is used as the production host, leveraging the availability of the iJN1462 genome scale model for P. putida KT2440 ¹¹ . Elementary mode analysis (EM A) ¹² is adapted to determine the constrained minimal cut set (cMCS) required to minimize metabolic flux towards undesired products and link indigoidine formation to cell viability ¹³ . These analyses combined with publicly available omics data ^{14 15} provide the set of gene loci that represented the reactions necessary for removal. The corresponding set of gene loci are repressed using multiplex CRISPR interference (CRISPRi) that is optimized for use in P. putida KT2440.

[0090] Such an implementation results in a highly edited strain that, in a single iteration of strain engineering, achieves close to 50% max theoretical yield of indigoidine in P. putida KT2440 and TRY characteristics that maintain fidelity from laboratory to industrially relevant scales. The specific aspects of our systems, the PrOSE approach, its implementation, and other beneficial characteristics demonstrated by the PrOSE strain are described in this study.

RESULTS

Genome scale evaluation of P. putida for production-obligatory growth

[0091] To develop the PrOSE approach (Figure 1A), it is first explored how many of the represented metabolites in P. putida ΪTN11462 ¹¹ model can be made obligatory for growth.

For this the cMCS algorithm ¹³ is used to identify minimal sets of reactions, the elimination of which would cause production of a given metabolite to become essential for growth. Aerobic conditions with glucose as the sole carbon source are used to model growth parameters. The theoretical yield is Yproduct/Substrate, hereafter referred to as YP/S. Gene knockdown sets are searched to satisfy three potential constraints in which YP/S is at minimum, 10%, 50%, or 80% of the maximum theoretical yield (MTY) for all producible metabolites in the model. This analysis is completed for all 2145 metabolites in the genome scale model, which indicates that 979 organic metabolites could be potentially growth coupled using under these growth conditions. In the first pass, 95.7% of all metabolites had the potential to be obligatory for growth, with a minimum threshold of 10% MTY (Table 2); this is consistent for similar calculation for other hosts ¹³ . When the threshold for minimum production is set to 50% MTY, 950 metabolites could be obligatory for growth; for an 80% threshold MTY, only 343 metabolites could be obligatory for growth, representing only 35% of the total producible metabolites. Thus setting the higher the threshold for minimum product yield results in fewer metabolites that can be used to implement a production obligatory regime. [0092] Table 2.

¹ Results for E. coli were reported previously (von Kamp and Klamt 2017)

² 892 unique metabolites

³ 95.7%

⁴ Metabolites include 4-Hydroxy-L-threonine, Mannose-6-phosphate, Mannuronate EOnly one metabolite - Vaccenyf coenzyme A

[0093] An in silico reaction for the heterologous product, indigoidine, is added to the genome scale metabolic model iJN1462 ¹¹ . This reaction represents the biosynthesis of indigoidine from glutamine and accounts for all cofactors needed. YP/S for glutamine and indigoidine is calculated to be 1.141 mol/mol and 0.537 mol/mol respectively from glucose as the carbon source (Table 1). YP/S for glutamine in P. putida is relatively high relative to other hosts screened (Table 3). As this method accounts for the other physiological processes competing for resources, a YP/S derived from a genome scale model provides a more accurate assessment compared to simpler methods, as is commonly done in the field ^{16 17} .

[0094] Table 1. Maximum theoretical yield of glutamine and indigoidine from three different substrates Glucose, Galactose and p-coumarate with respect to stoichiometry and redox balance in P. putida

[0095] Table 3. Theoretical yields of glutamine and indigoidine across different industrial hosts.

[0096] In order to predict reactions that would be required to improve indigoidine production, glutamine, its precursor, is used to conduct the analysis. The process for determining the list of required gene targets is diagrammed in Figure 1A. The minimum YP/S of glutamine is set at 10%, 50% and 80% to derive the reactions that would require knockout or knockdown for product obligatory growth. Potential target sets which required the removal of multi-functional gene products is eliminated, as it is sought to limit additional metabolic perturbations that could confound our analysis. Of the 1956 reactions in iJN1462 that are associated with genes, only 60% have a single gene associated with them. If a metabolic reaction is catalyzed by more than one gene (i.e., gene redundancy), both genes are included for inactivation. After implementing these filters, it is found that a threshold of 80% YP/S could be achieved using the elimination of 14 cellular reactions. These 14 metabolic reactions when mapped to their corresponding genes and gene products represent 16 gene loci (Figure IB)

[0097] Next, using Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA), it is confirmed the 16 gene cMCS strategy to be obligatory for glutamine production. Using the constructed cMCS platform, the parameters are set to explore potential product-obligatory strategies to enhance the production of indigoidine in P. putida when fed glucose as the sole carbon source. The focus is narrowed to a minimum glutamine production of 80% YP/S and a minimum biomass yield of 0.01 gDW mmol ¹ . This 16 gene set designed for glutamine is then extended to assess production obligatory growth for indigoidine. It is found that indigoidine production is obligatory with 90% theoretical yield of indigoidine (0.48 mol/mol glucose). This workflow allowed one to adapt the work from von Kamp and Klamt for non native final products and predict specific genes rather than enzymatic reactions for knockdown.

[0098] To test the flexibility of this PrOSE strategy, the substrate utilization range is expanded to better match sugars found in emerging lignocellulosic hydrolysates. An analysis of suitable starting carbon sources indicated that cultivation with several amino acids or sugars (i.e. lysine, pCA) failed to produce glutamine (Table 4). P. putida can be engineered to express the minimal E. coli galETKM utilization pathway ^18,19 . Interestingly, this gene targeting set results in production obligatory growth using galactose as a carbon source because both sugars share the same downstream catabolism (Figure IB).

[0099] Table 4: An analysis of suitable starting carbon sources indicated that cultivation with several amino acids or sugars (i.e. lysine, pCA) failed to produce glutamine.

Building the PrOSE strain:

[00100] To test the predictions from the metabolic modeling described above, the control and PrOSE P. putida system are built. First the heterologous production pathway is genomically integrated. The pathway for the indigoidine is comprised of sfp and bpsa. BpsA is a non-ribosomal peptide synthetase (NRPS) from Streptomyces lavendulae that catalyzes indigoidine formation from 2 molecules of glutamine in an ATP-dependent manner ²⁰ . Activation of BpsA requires a post-translational pantetheinylation via a promiscuous Sfp from Bacillus subtilis. The genomically integrated production strain harboring a plasmid- borne dCpfl and non-targeting gRNA serves as the control strain. The basal production of Indigoidine in P. putida is 2.3 g/L indigoidine from 10 g/L glucose after 24 hours (Figure 4D). The bulk of production occurs in the stationary phase, approximately 12 hours after carbon depletion. To test the use of galactose, a galactose utilization strain is also engineered via genomic integration of a galETKM operon ^18,19 and here production of indigoidine is negligible (Figures 4A to 4E). Optimizing carbon/nitrogen ratio yields only modest improvements to indigoidine production for both glucose and ammonium sulfate (Figures 1A to 4C).

[00101] Prior to construction of the PrOSE strain, the gene set is assessed to determine whether it contains any essential genes. The iJTST1462 model has an incomplete list of essential genes; in addition genes as essential or dispensable are manually annotated using gene essentiality data generated from a barcoded fitness library (RB-TnSeq) ¹⁵ . Two genes are found to be essential. The remaining 14 genes are targeted for knockdown using a multiplex CRISPRi/dCpfl targeting array. Based on the understanding of repetitive element instability ^21,22 use of repeated DNA sequences is minimized to limit gRNA array loss. While other reports have indicated technical challenges constructing multiplex gRNA arrays ²³ , both native and synthetic repetitive arrays exist (including those of native CRISPR arrays) ^24,25 . An endonuclease deficient class II CRISPR-Cas enzyme, Cpfl, is chosen over Cas9 as the Cpfl crRNA is only 19 bp in size, compared to the corresponding crRNA (gRNA scaffold sequence) from Cas9, which is 76 bp ²⁶ . Each gRNA is driven by a different P. putida tRNA ligase promoter/terminator pair and dCpfl is placed under the control of the lacUV5 promoter. Minimal 100 bp promoter sequences from native tRNA ligases are sufficient to express mCherry fluorescent protein, confirming that heterologous mRNA transcripts for gRNAs would be generated (Figure 4E).

[00102] In a successful deployment of the multiplex CRISPRi/dCpfl a decrease in mRNA expression levels (and protein abundance) is expected of the genes targeted with CRISPR interference. RNAseq analysis is used to examine the PrOSE strain, and compare normalized RNA expression levels between the control strain to a production strain carrying the glucose-PrOSE targeting plasmid (Figures 2A and 2B). RNA expression levels are sampled over the duration of a 72 hour time course. Expression of all 14 gRNAs are detected by this analysis. Expression of gRNAs is highest using the synthetic J23101 promoter but all native tRNA ligase promoters driving the remaining gRNAs in the array are functional. The multiplexed Cpfl gRNAs in this array does not efficiently terminate with endogenous terminator sequences, and generated chimeric mRNAs are generated. Nonetheless, eight of the fourteen targeted gene loci exhibit decreased mRNA expression levels, but at best show a 50% decrease. Gene knockdown efficiency of the same samples is confirmed using high resolution proteomics and indicate a similar reduction of protein abundance (Figure 2B). Despite the partial knockdown, growth assays show limited growth on lysine as carbon source, per the predictions from the FBA modeling (Figure 2D).

Characterizing the ProSE strain

[00103] A successful redirection metabolic flux to form glutamine, results in the PrOSE strain possessing several new quantifiable changes relative to the control. High TRY for the desired product is expected since more YP/S glutamine should result in more indigoidine. The production of indigoidine would shift from stationary phase to exponential phase, as the cell must form glutamine/indigoidine to survive. And finally, these phenotypes should maintain fidelity across a range of growth modes and scales.

[00104] Whether indigoidine production is improved in the PrOSE relative to the control strains is tested in several laboratory cultivation formats. Production for both the native glucose carbon source as well as galactose are tested. The PrOSE and a control strain are cultivated with either 56mM glucose or 56mM galactose, as the same product obligatory targeted set would function on either carbon source. In a deep well plate format, it is observed that the PrOSE strain produce nearly three-fold more indigoidine than the control strain when fed glucose (Figures 3A to 3C). In a shake flask format, the PrOSE strain produces 30% more than the control strain. Finally, when cells are cultivated with galactose in the deep well format, the same PrOSE strain is able to produce indigoidine in contrast to the gal utilization control strain which only forms biomass.

[00105] In a batch-mode bioreactor, improved titers for indigoidine are observed when cells are cultivated with glucose as the carbon source. In this format, the PrOSE strain produces 12.5 g/L indigoidine from 60 g/L glucose. The control production strain, in contrast, produces 5 g/L, and production of the final molecule is still realized after glucose is exhausted from the media. When fed galactose, the PrOSE strain also shows improved titers under batch-mode conditions. The PrOSE strain successfully produces 25.6 g/L of indigoidine from 60 g/L galactose, while the control strain produces around 900 mg/L of indigoidine, a 28 fold improvement. Moving to an industrially relevant cultivation format did not impact final product titer, allowing further development of fed-batch cultivation methods with these strains.

[00106] Using a fed-batch cultivation regime realizes greater improvements in final product titer as well as production kinetics. After an initial high nutrient feed to increase biomass in the reactor, a reduced feed rate allows detection of indigoidine product formation during exponential phase growth. This observation fulfils the hypothesis which predicts that indigoidine formation must occur during exponential phase in order for the cell to survive. In terms of yield, the PrOSE strain shows consistently higher production than the control strain when cultivated with glucose, but is not as consistent when cultivated on galactose. Together all aspects of the phenotypes that are desirable for the PrOSE are found to be true. DISCUSSION

[00107] The competition between biomass accumulation and production of the target compound is a well recognized challenge. This trade-off impacts both TRY and scalability. Approaches to address this tradeoff range from growth coupling ²⁷ to growth decoupling ²⁸ .

[00108] Canonical examples of growth coupling include FBA-based methods such OptKnock ²⁹ , a well-used approach that identifies secondary pathways that reduce the pool of a key intermediate as means to increase flux to the target of interest. This strategy has been described as weak growth coupling ³⁰ where growth still occurs even if the desired product is not being formed. Genomatica used weak growth coupling to improve 1,3 BDO production in E. coli to 18 g/L, but their strain engineering required a suite of additional refinements including adaptive lab evolution and bottleneck metabolite analysis ³¹ in order to implement at scale. Others have described growth coupling as the creation of a “driving force” such as ATP production or cofactor imbalance, and link the “driving force” to the desired production pathway ^27,32-34 . “Driving force” coupling is also pathway specific and requires additional strain engineering, such as for 1 -butanol production in E. coli using NADH as the driving force ³² or media supplementation for butanone production that is linked to acetate assimilation in E. coli ²⁷ .

[00109] In contrast to the examples described above, PrOSE is an implementation of strong growth coupling. It relies on EMA based methods that have been shown at genome scale level ^13,35 ’ ³⁶ but often suggests a large number of enzymatic reactions for elimination. FBA is used to validate the optimal cMCS and removes essential genes from targeted gene sets using -omics data to determine which genes should be targeted for CRISPRi. This work is the first experimental implementation of a genome-scale strong growth coupling solution set. Reliability in scaling is a necessary part of strain engineering that also remains unexplored in most cases. When pursued it often takes years. A successful implementation of PrOSE allows one to achieve in one cycle of strain engineering not only a very high TRY but also due to the inherent obligatory product formation, the production parameters are consistently maintained from batch to fed-batch and from microtiter plates to 2L bioreactors.

[00110] In the context of TRY improvement alone, indigoidine itself is an example of a heterologous product that has been demonstrated at high titers ^37-39 . The production of indigoidine is high in the oleaginous yeast Rhodosporidium toruloides but remains low in the model yeast S. cerevisiae, despite similar optimization of cultivation parameters. This comparison represents an empirical example of the innate metabolic potential of a given host, and is consistent with our calculated max theoretical yields for indigoidine (Table 3).

Genome scale metabolic models can accurately predict how microbial hosts could be advantageous for the production of a given metabolite. For indigoidine, the max YP/S from glucose in P. putida is 0.54 and is comparable to that fori? toruloides , while E. coli and S. cerevisiae are much lower. It is likely that every molecule will be different. Thus selecting the best host/ final product pair is a crucial aspect of developing the ideal production platform.

[00111] While this implementation of PrOSE is successful, several caveats of this approach are not addressed in this study. For indigoidine production in P. putida , the 90% maximum yield that is theoretically possible is not reached (whereas industrial yeast ethanol production is near 90% MTY). It is speculated that this is due to the incomplete knockdown on the gene set. With a better understanding of the terminator sequence efficiency (as observed in P. putida and E. coli ²³ ) more efficient CRISPR mediated gene knockdown may be achieved. Additional MFA analysis from 13C glucose or 13C galactose using these strains could also refine the genome scale models, enabling more accurate metabolic flux modeling when these engineered strains are grown with these carbon sources. PrOSE also relies on the availability of a high quality genome scale metabolic models, and product obligatory growth is calculated using a single carbon source. Thus currently, PrOSE cannot be used for certain mixed carbon streams, such as glucose and xylose, as our calculations for glucose-PrOSE inactivates the pentose phosphate pathway. Similarly, there are metabolites that cannot be made obligatory for growth ¹³ . Alternative strategies for final products derived from this class of metabolites would need to be explored. Products or intermediates that may be toxic are not considered. As final processes also use renewable carbon sources that may contain inhibitory byproducts, microbial hosts will require some degree of tolerance engineering ⁴⁰ to unlock its potential. Addressing these aspects will further boost the usefulness of PrOSE.

[00112] This approach is a significant step forward in realizing the full potential of microbial processes. Part of the appeal of genome scale models is that their genetic solutions are scale-agnostic; the predicted metabolic rewiring should apply even in the largest bioreactor formats. PrOSE is the first implementation of theoretical predictions for product obligatory metabolite production and is realized by multiplex CRISPR interference. By making the final desired product obligatory for growth, one is able to mimic native obligatory product formations such as ethanol production in budding yeast that have been so successful. Further, there are apparently no other examples in which production of a non-native molecule is deliberately shifted from stationary phase to exponential phase as a result of strain engineering. This holistic approach represents a new paradigm for the evaluation and optimization of microbial host / final product pairs.

[00113] The optimization of final product titer, rate, and yield during scale-up is arguably the most critical step required to support the field of biotechnology; it is also the most laborious and time consuming. For example, the heterologous production of isopentenol in E. coli has been improved from trace concentrations to >2g/L over a decade of experimental work ^41,42 . PrOSE provides a powerful new approach to significantly reduce the time taken to achieve these high production parameters, and inherently designs a strain that maintains its production parameters across scales.

MATERIALS AND METHODS

Computation of constrained minimal cut sets (cMCS)

[00114] Pseudomonas putida KT2440 genome scale metabolic model (GSM) iJN1462 ^u is used. The ATP maintenance demand and glucose uptake are 0.97 mmol ATP/gDW/h and 6.3 mmol glucose/gDW/h respectively. Constrained minimal cut sets (cMCS) are calculated according to the algorithm as previously described ¹³ . Excretion of byproducts is initially set to zero, except for the reported overflow metabolites for secreted products specific to P. putida (gluconate, 2 ketogluconate, 3 oxoadipate, catechol, lactate, methanol, CO2, and acetate). Additional inputs including minimum demanded product yield (% of the maximum product yield) and minimum demanded biomass yield are also specified in order to constrain the desired design space. Knockouts of export reactions and spontaneous reactions are not allowed. The algorithm computes for all minimal combinations of reaction knockouts blocking all undesired flux distributions and maintaining at least one of the desired metabolic flux distributions. With the specifications used herein each calculated knockout strategy (cMCS) ensures that growth is not feasible without biosynthesis of glutamine. (Growth production locking/strong growth coupling/Product obligatory growth). All cMCS calculations are done using API functions of CellNetAnalyzer ⁴³ on MATLAB 2017b platform using CPLEX 12.8 as the MILP solver. Once all the cMCS are enumerated, the PrOSE workflow (Figure 1A) is used to find the best engineering strategy to carry forward.

Constraint Based methods to confirm the cMCS

[00115] i JN1462 is extended to account for indigoidine biosynthesis pathway and checked for strong growth coupling to confirm the chosen engineering strategy for experimental implementation. Flux Balance Analysis (FBA) is used to calculate the maximum theoretical yield (MTY) from reaction stoichiometry and redox balance and for single gene deletion analysis. Flux variability analysis (FVA) along with FBA is used to check for minimum and maximum glutamine or indigoidine flux under the identified cMCS strategy to confirm product obligatory growth. COBRA Toolbox v.3.0 ⁴⁴ in MATLAB R2017b is used for FBA and FVA simulations with the GLPK (website for: gnu.org/software/glpk), an open-source linear optimization solver.

Chemicals, media and culture conditions

[00116] All chemicals and reagents are purchased from Sigma-Aldrich (St. Louis, MO) unless mentioned otherwise. When cells are cultivated in a microtiter dish format, plates are sealed with a gas-permeable film (Sigma-Aldrich, St. Louis, MO). Tryptone and yeast extract are purchased from BD Biosciences (Franklin Lakes, NJ). Engineered strains are grown on M9 Minimal Media as described previously ⁴⁵ with slight modifications. Carbon sources (glucose, p-coumarate or galactose) are used at 10g/L and (NH ₄ ) ₂ SO ₄ is used at 2 g/L.

Strains and strain construction

[00117] Pseudomonas putida KT2440 is used as the host for strain engineering.

Electroporation with the respective plasmid is performed using BIORAD MicroPulser preprogrammed EC2 setting (0.2 cm cuvettes with 100μL cells, ~5msec pulse and 2.5kV). Transformed cells are allowed to recover at 25 C for around 2.5 hours followed by plating onto selective media (containing respective antibiotics) and overnight incubation. Positive clones are confirmed for the genotype by colony PCR using Q5 Polymerase enzyme (NEB, Ipswitch, MA). The dCpfl/CRISPRi system ²⁶ is adapted for use in P. putida by subcloning dCpfl into a pBBRl backbone and placed under the LacUV5 promoter. The synthetic gRNA array is constructed using gene synthesis techniques (Genscript, Piscataway, NJ) and cloned into the dCpfl/CRISPRi backbone using isothermal DNA assembly. All plasmid constructs are verified with Sanger sequencing before transformation into Pseudomonas putida. Analytics/ Sugar Quantification - HPLC

[00118] Glucose and organic acids from cell cultures are measured by an 1100 Series HPLC system equipped with a 1200 Series refractive index detector (RID) (Agilent) and Aminex HPX-87H ion-exclusion column (300 mm length, 7.8 mm internal diameter; Bio- Rad Laboratories, Inc., Hercules, CA). Three hundred-microliter aliquots of cell cultures are removed at various time points during production and filtered through a spin-cartridge with a 0.45-pm nylon membrane, and 10 pL of the filtrate is eluted through the column at 50°C with 4 mM sulfuric acid at a flow rate of 600 pL/min for 30 min. Metabolites are quantified by using external standard calibration with authentic standards.

Indigoidine Quantification

[00119] Briefly, lOOuL of cells is pelleted by centrifugation at 15000 rpm for 2 min. The supernatant is discarded and 500uL DMSO is added to the pellet. The solution is vortexed vigorously for 30 s to dissolve Indigoidine. After centrifugation at 15000 rpm for 2 min, 100pL of DMSO extracted indigoidine is added to 96-well flat- bottomed microplates. Indigoidine is quantified by measuring the optical density at 612 nm wavelength (OD612) using a microplate reader (Molecular Devices Spectramax M2E) preheated to 25°C and applying standard curve generated from indigoidine purified from P. putida (Figure 5). Equation of Y (g/L of Indigoidine) = 0.212*A ₆₁₂ - 0.0035. The purity of extracted indigoidine from both E.coli and P. putida are cross-validated by NMR (method) (Figure 6).

RNAseq and data analyses

[00120] Total RNA is prepared following standard laboratory protocols ⁴⁶ for Trizol RNA extraction with several modifications. RNA from trizol treated lysates are bound to a silica column (Direct-zol RNA MiniPrep Plus Kit, Zymo Research, Irvine, CA) and its integrity confirmed using a Bioanalyzer RNA 6000 Nano assay (Agilent Technologies, Santa Clara, CA). Preparation of total RNA for RNAseq analysis is conducted at the DOE Joint Genome Institute on an Illumina MiSeq with 300bp paired end reads. Reads are assembled to the reference genome using BowTie with the following parameters. Reported gene expression values are the total normalized transcripts per million (TPM).

Targeted proteomics

[00121] Targeted proteomics is conducted as previously described ⁴⁷ . C ultivation at different scales

[00122] Cultures from glycerol stocks are struck to single colonies on LB agar media with the appropriate antibiotic as necessary. Single colonies are used to inoculate overnight cultures in LB with the appropriate antibiotic. Saturated overnight LB cultures are then back- diluted 1/lOOx into M9 minimal media with the appropriate carbon source as indicated. Cultures are back-diluted and adapted twice to ensure robust cell growth before heterologous pathway induction. All cultures are incubated with shaking at 200 rpm and 30°C. To prepare cells for pathway induction, M9 adapted cultures are back-diluted to a starting OD600 of 0.1, at which point IPTG and arabinose are added as appropriate. Production cultures are grown in 24 well deep well plates inoculated into a 200 uL culture volume and incubated InFors Multitron HT Double Stack Incubator Shaker set to 999 rpm linear shaker, and 70% humidity. For shake flask experiments, 60 mL cultures are grown in 250mL unbaffled Erlenmeyer shake flask and incubated at 200 rpm with orbital shaking. For all experiments, the indigoidine pathway is induced with 0.3% w/v L-arabinose, and dCpfl mediated gene repression is induced with 500 mM IPTG. Indigoidine production titers are also analyzed in Lysogeny-Broth (LB) containing 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl.

Fed-batch experiments at 2 L bioreactor scale

[00123] Fed-batch experiments are performed at 2 L scale using a Sartorius BIOSTAT B® fermentation controller, fitted with two Rushton impellers fixed at 800rpm. Initial reactor volume of 1L M9 Minimal Media (lOg/L Glucose, 0.3% w/v L-arabinose, 30mM NH4+), and 50mL overnight pre-culture in the same media. Cultivation temperature held at 30°C. Bioreactor processes are maintained to pH 7 using 10M NaOH.

Ambr Method: 250 mL ambr250™ bioreactor cultivations

[00124] Bioreactor experiments are carried out in a 12-way ambr250 bioreactor system (Sartorius Stedim Biotech GmbH, Goettingen, Germany) equipped with 250 mL single-use, disposable bioreactors (Microbial vessel type). The vessels are filled with 150 mL M9 minimal salt media for experiments with glucose as carbon source. Temperature is maintained at 30 ^° C throughout the fermentation process and agitation is set constant to 1300 RPM. Airflow is set constant to 0.5 VVM based on respective initial working volumes and pH is maintained at 7 using 4 N NaOH. Reactors are inoculated manually with pipettes with 5 mL of cell suspension grown under seed culture. After an initial batch phase of 12 hours, cultures with glucose as carbon source are fed with a concentrated glucose feed solution (600 g/L glucose ; Feed solution A) using two separate feed strategies: continuous feeding for one strain and pulsed feeding for another strain. For the continuous feed, the initial feed delivery is maintained at 5 g _gic /Lh or 1.292 mL/h. Pulsed feed is administered every two hours in the form of boluses restoring glucose concentrations to 10 g/L (feed parameters: 3.1 min @ 50 mL/h). After observing glucose accumulation, feed addition is paused for both conditions. Feed is resumed when glucose levels dropped below 10 g/L at reduced feed rates: 1 g _glc /Lh or 0.258 mL/h (continuous feed) and 3.23 g _gic /L or 1 min @ 50 mL/h (pulse feed). Similarly, during the fed-batch stage, recurring carbon exhaustion events are observed by DO signal spikes that triggered feed addition (Feeding parameters: ADO = 15 %, 10 min @ 33 mL/h). Samples are taken once every day (2 mL) and stored at -20 ^° C. The ambr250 runtime software is used to execute all process steps unless stated otherwise.

[00125] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.