Compositions and methods for biomolecule synthesis

Abstract

Provided herein are, inter alia, are cell compostions and methods for the biosynthesis of biomolecules.

Description

COMPOSITIONS AND METHODS FOR BIOMOLECULE SYNTHESISRELATED APPLICATION DATA

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Patent Application No. 63 / 583,543, filed on September 18, 2023, which is hereby incorporated by reference in its entirety and for all purposes.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under GM149120 and GM085770, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] In the face of global environmental challenges and limited fossil fuels, developing sustainable alternatives to petroleum-based production of chemicals and materials is a top priority. Microbial bioproduction offers a promising solution. Industrial microbial fermentation has been used for decades to produce an array of valuable organic compounds, such as antibiotics, vitamins, and food ingredients that were historically limited to endogenous products. Modern synthetic biology tools now make it possible to bioengineer microbes to produce a wide variety of heterologous (microbe, plant, animal) and new-to-nature products, greatly expanding the biomanufacturing landscape to fine and bulk chemicals. Many of these efforts, however, initially achieve only detectable amounts of product before metabolic engineering workflows, which rely on iterative design-build-test-1 earn cycles, are employed to optimize production titers. This powerful approach is, however, labor, resource, and time intensive. Automated screening platforms can accelerate bioengineering workflows but require specialized equipment and occupy large footprints. New bioengineering approaches that are efficient and generic are needed. Xanthommatin (Xa) is naturally produced by arthopods and cephalopods and is of increased interest in materials chemistry as a natural, broad-spectrum dye. Xa is normally produced via chemical synthesis and there are no known sustainable microbial production methods for Xa. Provided herein, inter alia, are compositions and methods of use thereof to address these and other problems in the art.BRIEF SUMMARY

[0004] In an aspect is provided a bacterial cell including a formate-tetrahydrofolate(THF) ligase (Ft£L) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product, and wherein the bacterial cell endogenously produces a 1 -carbon (Cl) metabolite.

[0005] In another aspect is provided a bacterial cell including a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product, and wherein the bacterial cell endogenously produces a formate.

[0006] In another aspect is provided a bacterial cell including a xanthommatin (Xa).

[0007] In an aspect is provided a bacterial cell including a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product, and wherein the bacterial cell endogenously produces a 1 -carbon (Cl) metabolite.

[0008] In another aspect is provided a cell, wherein the cell includes an auxotroph.

[0009] In another aspect is provided a method for synthesizing a biomolecule, the method including: (a) culturing the cell provided herein including embodiments thereof; and (b) allowing the cell to synthesize the biomolecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows an overview of an exemplary Cl auxotroph system. Deletions in serine hydroxymethyltransferase (glyA) and enzymes of the glycine cleavage system gcvTHP block biosynthesis of the essential cofactor 5,10-methylene-THF (MTHF). Biosynthesis of MTHF is restored via a heterologous formate assimilation pathway from Methylobacterium extorquens, consisting of the formate-THF ligase (FtfL), methenyl-THF cyclohydrolase (FchA), and methylene- THF dehydrogenase (MtdA).

[0011] FIG. 2 shows a schematic of Cl -based growth-coupled biosynthesis. The biosynthetic pathway releases formate, which links pathway activity to growth via biosynthesis of the essential cofactor 5, 10-methylene-THF.

[0012] FIG. 3 shows the release of formate by KFA directs metabolic flux towards production of target compounds.

[0013] FIG. 4 shows a schematic of xanthommatin (Xa) biosynthesis.

[0014] FIG. 5 shows a schematic of the growth-coupling strategy described herein.

[0015] FIG. 6 shows the design and construction of a formate auxotroph described herein.

[0016] FIG. 7 shows exogenous formate rescues growth in a formate auxotroph described herein.

[0017] FIG. 8 shows formate concentration dependence of an auxotroph described herein.

[0018] FIG. 9 shows formate consumption is coupled to growth in a formate auxotroph described herein.

[0019] FIG. 10 shows exemplary heterologous pathways for production of KYN and 3-HK.

[0020] FIGS. 11A-11B show results from an experiment demonstrating that the heterologous pathway rescues growth of a formate auxotroph (FA) described herein.

[0021] FIGS. 12A-12B show tryptophane concentration dependence of a formate auxotroph described herein.

[0022] FIG. 13 shows production in a formate auxotroph (FA) described herein compared to a wildtype EM42.

[0023] FIGS. 14A-14F show exometabolite analysis from a formate auxotroph described herein.

[0024] FIG. 15 shows a schematic of an exemplary biosynthesis pathway described herein.

[0025] FIGS. 16A-16C show results from an evolution away from L-tryptophan feeding experiment.

[0026] FIGS. 17A-17C show exemplary systems for heterologous production designs. FIG. 17A: Schematic comparison of traditional (top) versus growth-coupled (bottom) heterologous production designs. In the complementary growth-coupled design, an auxotrophy is engineered for complementation with a heterologous metabolic byproduct that directly connects cell growth with product biosynthesis. FIG. 17B: Examples of animals that produce ommochromes / xanthommatin. FIG. 17C: The xanthommatin biosynthetic pathway in Drosophila melanogaster .

[0027] FIGS. 18A-18C show design (FIG. 18A) and confirmation (FIGS. 18B-18C) of theputida formate auxotroph. FIG. 18A: Model of growth-coupled design. Native glyA genes, encoding serine-hydroxyl-methyltransferase, and gcvTHP genes, encoding enzymes of the glycine cleavage system, are deleted, blocking biosynthesis of 5,10-methylene-THF. Heterologous genes ftfL, fdhA, and mtdA are introduced for synthesis of 5,10-methylene-THF from formate. FIG. 18B: Growth assay of strain LBB05 cultivated in minimal media with the supplements in each case [20 mM glucose (glc). 10 mM glycine (gly) and 10 mM formate]. Inset shows calculated growth rate. FIG. 18C: Growth assay of LBB05 cultivated in minimal media containing 20 mM glucose, 10 mM glycine, and formate at the concentration indicated. Inset shows calculated growth rate.

[0028] FIGS. 19A-19H show growth-coupled biosynthesis of kynurenine products in the P. putida formate auxotroph. FIG. 19A: Three-step biosynthetic pathway converting tryptophan to 3- hydroxy kynurenine (3HK) with formate as byproduct. FIG. 19B: Design of the kynurenine (KYN) and 3HK expression plasmids. Tryptophan supports the growth of P. putida strains LBB06 and LBB07, but not LBB05, in the absence of formate. FIG. 19D: Growth of P. putida strains LBB06 and LBB07 in minimal media is dependent on concentration of tryptophan. Increasing concentration of tryptophan results in faster growth rate and higher maximal OD600. FIG. 19D: Reversed-phase HPLC analysis of P. putida strains LBB06 VS. LBB11 and LBB07 vs. LBB12 and the production of 3HK, KYN, xanthurenic acid (XANA), kynurenic acid (KYNA), xanthommatin (Xa), and decarboxylated Xa FIG. 19E: Titers of exogenous metabolites achieved at 72 h of growth. FIG.19F: UV-vis absorbance spectrum of Xa. FIG. 19G: Culture flasks of LBB07 and LBB12 at 72 h of growth. FIG. 19H: Three-step biosynthetic pathway converting tryptophan to 3 -hydroxy kynurenine (3HK) with formate as byproduct;.

[0029] FIGS. 20A-20I show Adaptive laboratory evolution to engineer production from glucose only. FIG. 20A: Schematic of ALE protocol to wean strain off supplement. Strains were initially grown in media supplemented with growth-promoting glycine or tryptophan. After a fixed time in stationary phase, aliquots of the culture were propagated to both a batch containing a lower concentration of the growth-promoting agent and a “test” batch without the agent. The test flasks were propagated if growth was observed, and the adaptation was considered successful once at least two consecutive batches showed growth. FIG. 20B: Representative growth curves for the glycine adaption. FIG. 20C: Growth curves of single isolates from evolved populations. FIG. 20D: Worflow to investigate diversity of MetK mutations in evolved population. FIG. 20E: Frequency of a given mutation found in the population. FIG. 20F: Representative growth curve of the qbsFH Tryptophan adaptation. FIG. 20G: Representative growth curve of the qbsFGH Tryptophan adaption. FIGS. 20H-20I: Mutant plasmids were re-engineered into the original Cl auxotroph to demonstrate growth in tryptophan-free minimal medium.

[0030] FIGS. 21A-21C show bioproduction of final evolved strain. FIG. 21A: One of the glycine / tryptophan isolates was grown for 72 h in shake flasks and analyzed for production of metabolites over time. The decrease in 3-HK corresponds with an increase in Xa. FIG. 21B: representative color changes of the final evolved strain. FIG. 21C: Schematic of purification process. Reduced Xa precipitates from aqueous solution.

[0031] FIGS. 22A-22C show metabolic modeling for exemplary auxotrophs described herein. FIGS. 22A-22B: Flux space plots for the metabolic models. FIG. 22C: Metabolix flux balance analysis of model showing how formate is incorporated.

[0032] FIG. 23 shows bioproduction assays in minimal medium containing L-Trp (5 mM) with strains LBB06, LBB11, LBB07, and LBB12.

[0033] FIGS. 24A-24B show identification of xanthommatin by high-performance liquid chromatography (HLPC)-quadruple time-of-flight mass spectrometry (QTOF).

[0034] FIG 25 shows redox-dependent changes in wavelength absorbance of exemplary xanthommatin compounds described herein. Addition of chemical reductant ascorbic acid results ina shift in absorbance to shorter wavelengths of light. Addition of chemical oxidant sodium nitrite results in a shift in absorbance to longer wavelengths of light.

[0035] FIGS. 26A-26H show growth curves for the glycine auxotroph removal ALE experiment. All replicates showed consistent growth on the base media after a single batch of growth in glycine- supplemented media. Clonal isolates for all replicates were isolated from the last batch of growth in the non-supplemented media.

[0036] FIGS. 27A-27J show growth curves for the tryptophan auxotroph removal ALE experiment. All replicates showed consistent growth on the base media after no more than three batches of growth in tryptophan-supplemented media. Clonal isolates for all replicates were isolated from the last batch of growth in the non-supplemented media.

[0037] FIG. 28 shows growth rates of isolated evolved LBB09 (top panel) and LBB10 (bottom panel) clones for Tryptophan dependence removal.

[0038] FIGS. 29A-29D show bioproduction assays in shake flasks in minimal medium with glucose only, no supplements with LBB11 (FIG. 29A), LBB06 (FIG. 29B), LBB07 (FIG. 29C), and LBB08 (FIG. 29D).

[0039] FIG. 30 shows growth curves of P. putida LBB08 containing vanAB expression plasmid (gray) or gfp expression plasmid (black) in the presence of 1 mM vanillic acid in dB media + glucose. The strain expressing vanAB is able to rescue growth in the presence of vanillic acid. VA = vanillic acid, PCA = protocatechuic acid.

[0040] FIG. 31 shows a schematic of compounds that can be assimilated into the formate pathway and the enzymes that catalyze their conversion. Bacterial cells described herein may include one or more of the enzymes in the formate assimilation pathway.

[0041] FIG. 32 shows a schematic of compounds and enzymes in the kynurenine pathway. Bacterial cells described herein may produce one or more of the compounds in the kynurenine pathway. In addition, bacterial cells described herein may include one or more of the enzymes in the kynurenine pathway.DETAILED DESCRIPTIONDEFINITIONS

[0042] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0043] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0044] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0045] It should be noted that throughout the application that alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.

[0046] “ Analog,” “analogue,” or “derivative” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound.

[0047] The terms "a" or "an," as used in herein means one or more. In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl orheteroaryl group, is "substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C1-C20 alkyls, and / or one or more unsubstituted 2 to 20 membered heteroalkyls.

[0048] Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R- substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R13substituents are present, each R13substituent may be distinguished as R13 A, R13 B, R13 c, R13 D, etc., wherein each of R13 A, R13 B, R13 c, R13 D, etc. is defined within the scope of the definition of R13and optionally differently. Where an R moiety, group, or substituent as disclosed herein is attached through the representation of a single bond and the R moiety, group, or substituent is oxo, a person having ordinary skill in the art will immediately recognize that the oxo is attached through a double bond in accordance with the normal rules of chemical valency.

[0049] Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and / or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds

[0050] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

[0051] The terms “bind” and “bound” as used herein is used in accordance with its plain and ordinary meaning and refers to the association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be bound, e.g., by covalent bond, linker (e.g. a first linker or second linker), or non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole- induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).

[0052] The term “capable of binding” as used herein refers to a moiety (e.g. a compound as described herein) that is able to measurably bind to a target (e.g., a NF-KB, a Toll-like receptor protein). In embodiments, where a moiety is capable of binding a target, the moiety is capable of binding with a Kd of less than about 10 pM, 5 pM, 1 pM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.

[0053] As used herein, the term "conjugated” when referring to two moi eties means the two moi eties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent. In embodiments, the two moieties are covalently bonded to each other (e.g. directly or through a covalently bonded intermediary). In embodiments, the two moieties are non-covalently bonded (e.g. through ionic bond(s), van der Waal’s bond(s) / interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).

[0054] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0055] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; ornucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and mini circle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0056] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0057] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribosephosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0058] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0059] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homologysearching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0060] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0061] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0062] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, aswell as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0063] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0064] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0065] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be considered when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no aminoacid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0066] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0067] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0068] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see, e.g., Creighton, Proteins (1984)).

[0069] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site www.ncbi.nlm.nih. ov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50- 100 amino acids or nucleotides in length.

[0070] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0071] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0072] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0073] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0074] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0075] A "ligand" refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor or antibody, antibody variant, antibody region or fragment thereof.

[0076] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0077] The term “formate-tetrahydrofolate ligase protein” or “FtfL protein” is used herein according to its plain ordinary meaning and refers to a ligase enzyme that catalyzes the following reaction:ATP + formate + tetrahydrofolate — > ADP + phosphate + 10-formyltetrahydrofolate.In embodiments, the term FtfL protein includes any of the recombinant or naturally-occurring forms of the formate-tetrahydrofolate ligase protein, or variants or homologs thereof that maintain FtfL activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to FtfL). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring FtfL protein, such as the FtfL . In embodiments, the FtfL protein is substantially identical to the protein identified by the UniProt reference number Q83WS0 or a variant or homolog having substantial identity thereto.

[0078] The term “methenyl-THF cyclohydrolase protein” or “Fch protein” is used herein according to its plain ordinary meaning and refers to a hydrolase enzyme that catalyzes the following reaction:10-formyltetrahydrofolate — 5,10-methenyltetrahydrofolate + H2O.In embodiments, the term Fch protein includes any of the recombinant or naturally-occurring forms of the methenyltetrahydrofolate cyclohydrolase protein, also known as methenyl -tetrahydrofol ate cyclohydrolase, or variants or homologs thereof that maintain Fch activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to Fch). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring Fch protein. In embodiments, the Fch protein is substantially identical to the protein identified by the UniProt reference number Q49135 or a variant or homolog having substantial identity thereto. The terms Fch protein and FchA protein are used interchangeably herein.

[0079] The term “methylene-THF dehydrogenase protein” or “MtdA protein” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction:5,10-methenyltetrahydrofolate + NADPH2 —> 5,10-methylenetetrahydrofolate + NADPH+.In embodiments, the term MtdA protein includes any of the recombinant or naturally-occurring forms of the methylenetetrahydroformate dehydrogenase protein, also known as methylenetetrahydroformate dehydrogenase, or variants or homologs thereof that maintain MtdA activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to MtdA). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring MtdA protein. In embodiments, the MtdA protein is substantially identical to the protein identified by the UniProt reference number P55818 or a variant or homolog having substantial identity thereto.

[0080] The term “formaldehyde dehydrogenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: formaldehyde + NAD++ H2O — formate + NADH + H+.In embodiments, the term formaldehyde dehydrogenase protein includes any of the recombinant or naturally-occurring forms of the formaldehyde dehydrogenase protein, or variants or homologs thereof that maintain formaldehyde dehydrogenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to formaldehyde dehydrogenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring formaldehyde dehydrogenase protein.

[0081] The term “formate dehydrogenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: formate + NAD+CO2+ NADH + H+.In embodiments, the term formate dehydrogenase protein includes any of the recombinant or naturally-occurring forms of the formate dehydrogenase protein, or variants or homologs thereof that maintain formate dehydrogenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to formate dehydrogenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring formate dehydrogenase protein.

[0082] The term “cyanide hydratase” is used herein according to its plain ordinary meaning and refers to a hydratase enzyme (also known as a hydrolyase enzyme) that catalyzes the following reaction: hydrogen cyanide + H2O — formamide.In embodiments, the term cyanide hydratase protein includes any of the recombinant or naturally- occurring forms of the cyanide hydratase protein, or variants or homologs thereof that maintaincyanide hydratase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to cyanide hydratase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring cyanide hydratase protein.

[0083] The term “cyanide dihydratase” is used herein according to its plain ordinary meaning and refers to a hydratase enzyme (also known as a hydrolyase enzyme) that catalyzes the following reaction: hydrogen cyanide + 2H2O — formate.In embodiments, the term cyanide dihydratase protein includes any of the recombinant or naturally- occurring forms of the cyanide dihydratase protein, or variants or homologs thereof that maintain cyanide dihydratase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to cyanide dihydratase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring cyanide dihydratase protein.

[0084] The term “formamidase” is used herein according to its plain ordinary meaning and refers to a hydrolase enzyme that catalyzes the following reaction: formamide + H2O — formate + NH3.In embodiments, the term formamidase protein includes any of the recombinant or naturally- occurring forms of the formamidase protein, or variants or homologs thereof that maintain formamidase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to formamidase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring formamidase protein.

[0085] The term “carbon monoxide dehydrogenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction:wherein A and AH2 are an electron donor or electron receiver. Non-limiting examples of electron donor or an electron receiver include: ferredoxin, NADP+, NADPH, and flavoprotein complexes (e.g., flavin adenine dinucleotide (FAD), a hydrogenase, etc.). In embodiments, the term carbon monoxide dehydrogenase protein includes any of the recombinant or naturally-occurring forms of the carbon monoxide dehydrogenase protein, or variants or homologs thereof that maintain carbon monoxide dehydrogenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to carbon monoxide dehydrogenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring carbon monoxide dehydrogenase protein.

[0086] The term “carbonic anhydrase” is used herein according to its plain ordinary meaning and refers to a metalloenzyme that catalyzes the following reaction: carbonic acid CO2 + H O.In embodiments, the term carbonic anhydrase protein includes any of the recombinant or naturally- occurring forms of the carbonic anhydrase protein, also known as carbonate dehydratase, or variants or homologs thereof that maintain carbonic anhydrase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to carbonic anhydrase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring carbonic anhydrase protein.

[0087] The term “methylamine dehydrogenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: methylamine + H2O + acceptor — > formaldehyde + NH3 + reduced acceptor.In embodiments, the term methylamine dehydrogenase protein includes any of the recombinant or naturally-occurring forms of the methylamine dehydrogenase protein, also known as amine dehydrogenase or primary-amine dehydrogenase, or variants or homologs thereof that maintain methylamine dehydrogenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to methylamine dehydrogenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring methylamine dehydrogenase protein.

[0088] The term “dichloromethane dehalogenase” is used herein according to its plain ordinary meaning and refers to a lyase enzyme that catalyzes the following reaction: dichloromethane + H2O — formaldehyde + 2 chloride.In embodiments, the term dichloromethane dehalogenase protein includes any of the recombinant or naturally-occurring forms of the methylamine dehydrogenase protein, also known as dichloromethane chloride-lyase, or variants or homologs thereof that maintain dichloromethane dehalogenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to dichloromethane dehalogenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring dichloromethane dehalogenase protein.

[0089] The term “methanethiol oxidase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: methanethiol + O2 + H2O formaldehyde + hydrogen sulfide + H2O2.In embodiments, the term methanethiol oxidase protein includes any of the recombinant or naturally-occurring forms of the methanethiol oxidase protein, or variants or homologs thereof that maintain methanethiol oxidase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to methanethiol oxidase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring methanethiol oxidase protein.

[0090] The term “methanesulfonate monooxygenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: methanesulfonate + NADH + H+— > formaldehyde + NAD++ sulfite + H2O.In embodiments, the term methanesulfonate monooxygenase protein includes any of the recombinant or naturally-occurring forms of the methanesulfonate monooxygenase protein, or variants or homologs thereof that maintain methanesulfonate monooxygenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to methanesulfonate monooxygenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring methanesulfonate monooxygenase protein.

[0091] The term “methanol dehydrogenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: methanol — formaldehyde + 2 electrons + 2H+.In embodiments, the term methanol dehydrogenase protein includes any of the recombinant or naturally-occurring forms of the methanol dehydrogenase protein, or variants or homologs thereof that maintain methanol dehydrogenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to methanol dehydrogenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring methanol dehydrogenase protein.

[0092] The term “methane monooxygenase” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction: methane + O2 + NADPH + H+— > methanol + NADP++ H2O.In embodiments, the term methane monooxygenase protein includes any of the recombinant or naturally-occurring forms of the methane monooxygenase protein, or variants or homologs thereof that maintain methane monooxygenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to methane monooxygenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring methane monooxygenase protein.

[0093] The term “tryptophan dioxygenase protein” or “TDO protein” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes the following reaction:L-tryptophan + O2 — ► N-formyl-L-kynurenine.In embodiments, the term TDO protein includes any of the recombinant or naturally-occurring forms of the tryptophan dioxygenase protein, also known as tryptophan 2, 3 -di oxygenase, or variants or homologs thereof that maintain TDO activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to TDO). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring TDO protein. In embodiments, the TDO protein is encoded by a QbsF gene. In embodiments, the TDO protein is substantially identical to the protein encoded by the QbsF gene identified by the Joint Genome Institute Integrated Microbial Genomes & Microbiomes (JGI IMG / M) accession number Ga0439711 01 3519002 3519856 or a variant or homolog having substantial identity thereto. In embodiments, the TDO protein is substantially identical to the protein identified by the UniProt reference number Q84HF6 or a variant or homolog having substantial identity thereto.

[0094] The term “kynurenine formamidase protein” or “KFA protein” is used herein according to its plain ordinary meaning and refers to a hydrolase enzyme that catalyzes the following reaction:N-formyl-L-kynurenine + H2O — formate + L-kynurenine.In embodiments, the term KFA protein includes any of the recombinant or naturally-occurring forms of the kynurenine formamidase protein, also known as arylformamidase, or variants or homologs thereof that maintain KFA activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to KFA). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring KFA protein. In embodiments, the KFA protein is encoded by a QbsH gene. In embodiments, the KFA protein is substantially identical to the protein encoded by the QbsH gene identified by the Joint Genome Institute Integrated Microbial Genomes & Microbiomes (JGI IMG / M) accession number Ga0439711 01 3521286 3521942 or a variant or homolog having substantial identity thereto. In embodiments, the KFA protein is substantially identical to the protein identified by the UniProt reference number Q84HF4 or a variant or homolog having substantial identity thereto.

[0095] The term “kynurenine monooxygenase protein” or “KMO protein” is used herein according to its plain ordinary meaning and refers to a hydroxylase enzyme that catalyzes the following reaction:L-kynurenine + NADPH + H++ O2 — 3-hydroxy-L-kynurenine + NADP++ H2O.In embodiments, the term KMO protein includes any of the recombinant or naturally-occurring forms of the kynurenine monooxygenase protein, also known as kynurenine 3 -monooxygenase, kynurenine hydroxylase, or variants or homologs thereof that maintain KMO activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to KFA). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring KMO protein. In embodiments, the KMO protein is encoded by a QbsG gene. In embodiments, the KMO protein is substantially identical to the protein encoded by the QbsG gene identified by the Joint Genome Institute Integrated Microbial Genomes & Microbiomes (JGI IMG / M) accession number Ga0439711 _01 3519880 3521265 or a variant or homolog having substantial identity thereto. Inembodiments, the KMO protein is substantially identical to the protein identified by the UniProt reference number Q84HF5 or a variant or homolog having substantial identity thereto.

[0096] The term “aromatic L-amino acid decarboxylase protein” or “AADC protein” is used herein according to its plain ordinary meaning and refers to a lyase enzyme that catalyzes the decarboxylation of an L -amino acid. In embodiments, the term AADC protein includes any of the recombinant or naturally-occurring forms of the aromatic L-amino acid decarboxylase protein, or variants or homologs thereof that maintain AADC activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to AADC). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring AADC protein. In embodiments, the AADC protein catalyzes the decarboxylation of a L-kynurenine or an L -3 -hydroxykynurenine. In embodiments, the AADC protein catalyzes the decarboxylation of a L-kynurenine. In embodiments, the AADC protein catalyzes the conversion of an L-kynurenine to a kynuramine. In embodiments, the AADC protein catalyzes the decarboxylation of an L-3-hydroxykynurenine. In embodiments, the AADC protein catalyzes the conversion of an L-3-hydroxykynurenine to a 3-hydroxykynuramine.

[0097] The term “kynurenine aminotransferase protein” or “KAT protein” is used herein according to its plain ordinary meaning and refers to an aminotransferase enzyme that catalyzes the transamination reaction of an amino acid. In embodiments, the amino acid is a L-kynurenine or an L- 3 -hydroxykynurenine. In embodiments, the term KAT protein includes any of the recombinant or naturally-occurring forms of the kynurenine aminotransferase protein, or variants or homologs thereof that maintain KAT activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to KAT). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring KAT protein. In embodiments, the KAT protein catalyzes the transamination of a L-kynurenine or an L-3 -hydroxykynurenine. In embodiments, the KAT protein catalyzes the transamination of a L-kynurenine. In embodiments, the KAT protein catalyzes the conversion of an L-kynurenine to a kynurenic acid. In embodiments, the KAT protein catalyzes the transamination ofan L-3-hydroxykynurenine. In embodiments, the KAT protein catalyzes the conversion of an L-3- hydroxykynurenine to a xanthurenic acid.

[0098] The term “kynureninase protein” or “KYNU protein” is used herein according to its plain ordinary meaning and refers to a hydrolase enzyme that catalyzes the cleavage of a hydrogen bond of an amino acid. In embodiments, the amino acid is an L-kynurenine or an L-3 -hydroxykynurenine. In embodiments, the term KYNU protein includes any of the recombinant or naturally-occurring forms of the kynureninase protein, also known as L-kynurenine hydrolase, or variants or homologs thereof that maintain KYNU activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to KYNU). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring KYNU protein. In embodiments, the KYNU protein catalyzes the cleavage of an L-kynurenine or an L-3 -hydroxykynurenine. In embodiments, the KYNU protein catalyzes the conversion of an L-kynurenine to an anthranilic acid. In embodiments, the KYNU protein catalyzes the conversion of an L-3-hydroxykynurenine to a 3-hydroxyanthranilic acid.

[0099] The term “3-hydroxyanthranilic acid oxidase protein” is used herein according to its plain ordinary meaning and refers to an oxidase enzyme that catalyzes an oxidation-reduction reaction of a 3-hydroxyanthranilic acid. In embodiments, the term 3-hydroxyanthranilic acid oxidase protein includes any of the recombinant or naturally-occurring forms of the 3-hydroxyanthranilic acid oxidase protein, or variants or homologs thereof that maintain 3-hydroxyanthranilic acid oxidase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to 3-hydroxyanthranilic acid oxidase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring 3-hydroxyanthranilic acid oxidase protein. In embodiments, the 3- hydroxyanthranilic acid oxidase protein catalyzes the conversion of a 3-hydroxyanthranilic acid to a quinolinic acid.

[0100] The term “3-hydroxyanthranilic acid oxygenase protein” is used herein according to its plain ordinary meaning and refers to an oxidoreductase enzyme that catalyzes an oxidationreduction reaction of a 3-hydroxyanthranilic acid. In embodiments, the term 3-hydroxyanthranilic acid oxygenase protein includes any of the recombinant or naturally-occurring forms of the 3- hydroxyanthranilic acid oxygenase protein, or variants or homologs thereof that maintain 3- hydroxyanthranilic acid oxygenase activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to 3-hydroxyanthranilic acid oxygenase). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring 3-hydroxyanthranilic acid oxygenase protein. In embodiments, the 3-hydroxyanthranilic acid oxygenase protein catalyzes the conversion of a 3-hydroxyanthranilic acid to a picolinic acid.

[0101] The term “xanthommatin” or “Xa” is used herein according to its plain ordinary meaning and refers to an ommochrome xanthommatin biopigment. In embodiments, the xanthommatin is also known as 1 l-(3-amino-3-carboxypropanoyl)-l-hydroxy-5-oxo-5H-pyrido[3,2-a]phenoxazine-3- carboxylic acid, or variants or homologs thereof. In embodiments, the Xa has the formula

[0102] The term “dihydroxanthommatin” is used herein according to its plain ordinary meaning and refers to a reduced xanthommatin compound. In embodiments, the dihydroxanthommatin has the formula:

[0103] The term “auxotroph” is used herein according to its plain ordinary meaning and refers to a mutant cell that does not produce a biomolecule through its native process and requires a particular biomolecule in order to grow and expand. In embodiments, the cell is incapable of synthesizing the biomolecule except under selective conditions. In embodiments, the selective conditions include exogenous supplementation of the biomolecule. In embodiments, the selective conditions include a heterologous or exogenous enzyme or cellular pathway that generates the biomolecule from a relevant substrate. In embodiments, the selective conditions are growth-coupling.

[0104] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0105] The term “glyA gene” is used herein according to its plain ordinary meaning and refers to a gene that encodes a serine hydroxymethyltransferase (SHMT) protein. In embodiments, the term glyA gene includes any of the recombinant or naturally-occurring forms of glyA, or variants or homologs thereof. In embodiments, the glyA gene encodes a glyA gene product. In embodiments, the glyA gene includes glyA-1. In embodiments, the g / jM gene includes glyA-Il. In embodiments, the glyA gene includes glyA-I and glyA-II.

[0106] The term '"glyA gene product” is used herein according to its plain ordinary meaning and refers to a protein encoded by ag / gene. In embodiments, the glyA gene product is a serine hydroxymethyltransferase (SHMT) protein.

[0107] The term “serine hydroxy methyltransferase” or “SHMT” is used herein according to its plain ordinary meaning and refers to a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the following conversions: (i) L-serine to glycine; and tetrahydrofolate to 5,10- methylenetetrahydrofolate. In embodiments, the term SHMT protein includes any of the recombinant or naturally-occurring forms of the serine hydroxymethyltransferase protein, or variants or homologs thereof that maintain SHMT activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to SHMT). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 25, 50, 100, or 150 continuous amino acid residues) compared to a naturally occurring SHMT protein.

[0108] The term “gcvTHP gene” is used herein according to its plain ordinary meaning and refers to a gene operon that includes genes encoding proteins of the glycine cleavage system. In embodiments, the term gcvTHP gene includes any of the recombinant or naturally-occurring forms of the gcvTHP group of genes, or variants or homologs thereof. In embodiments, the gcvTHP gene encodes a gcvTHP gene product. In embodiments, the gcvTHP gene includes a gcvTHP-I gene or a gcvTHP -II gene. In embodiments, the gcvTHP gene includes agcvTHP-I gene and agcvTHP-H gene. In embodiments, the gcvTHP gene includes a gcvTHP-I gene. In embodiments, the gcvTHP gene includes a gcvTHP-II gene. In embodiments, the gcvTHP gene includes a gcvT gene. In embodiments, the gcvT gene encodes a tetrahydrofolate-dependent aminometyltransferase, also known as a T-protein. In embodiments, the tetrahydrofolate-dependent aminometyltransferase catalyzes the transfer of a methylene carbon unit from a glycine to a tetrahydrofolate. In embodiments, the gcvTHP gene includes a gcvH gene. In embodiments, the gcvH gene encodes a glycine cleavage system H-protein. In embodiments, the glycine cleavage system H-protein is a cosubstrate and carries the amniomethyl intermediate and then hydrogen through the prostetic lipoyl moiety. In embodiments, the gcvTHP gene includes a gcvP gene. In embodiments, the gcvP gene encodes a pyridoxal phosphate-containing protein, also known as a P-protein. In embodiments, the pyridoxal phosphate-containing protein is a glycine dehydrogenase enzyme. In embodiments, the glycine dehydrogenase enzyme catalyzes the decarboxylation of glycine. In embodiments, the gcvTHP gene includes a gcvT gene, a gcvH gene, and a gcvP gene. In embodiments, the gcvTHPgene includes an Ipd gene. In embodiments, the Ipd gene encodes a lipoamide dehydrogenase. In embodiments, the gcvTHP gene includes a gcvT gene, a gcvH gene, a gcvP gene and an Ipd gene.

[0109] The term “gcvTHP gene product” is used herein according to its plain ordinary meaning and refers to a protein encoded by a gcvTHP gene. In embodiments, the gcvTHP gene product is an H-protein, a T-protein, or a P-protein. In embodiments,gcvTHP gene product is an H-protein, a T-protein, a P-protein, or an L-protein. In embodiments, the gcvTHP gene product is an H-protein. In embodiments, the gcvTHP gene product is a T-protein. In embodiments, the gcvTHP gene product is a P-protein. In embodiments, the gcvTHP gene product is an L-protein.

[0110] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0111] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral -based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non- viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms ''transfection'' or "transduction" also refer to introducing proteins into a cell from the external environment. Typically,transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1- 4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0112] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0113] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOTA, NOTA, NETA, TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups. The chelate is normally linked to the PSMA antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as223Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an A1-18F complex, to a targeting molecule for use in PET analysis.

[0114] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0115] The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a pharmaceutical composition as provided herein and a cell. In embodiments contacting includes, for example, allowing a pharmaceutical composition as described herein to interact with a cell.

[0116] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0117] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0118] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0119] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0120] The term "exogenous" refers to a molecule or substance (e g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. In embodiments, where a protein (e.g. an enzyme used in a synthetic process provided herein) is exogenous to a bacterial cell provided herein, the protein is expressed by the bacterial cell provided herein from an exogenous nucleic acid encoding the protein within the bacterial cell. In embodiments, the exogenous nucleic acid is provided to the bacterial cell provided herein through known bacterial cell engineering or transfection techniques (e.g. cationic pikymers, viral transfection or electroporation).

[0121] The term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism. The term “endogenously produced” or “endogenously produces” as used herein refers to production of a biomolecule within a bacterial cell provided herein. As described herein, an endogenously produced biomolecule may be naturally produced by the bacterial cell provided herein or may not be naturally produced by the bacterial cell provided herein. In embodiments, the endogenously produced biomolecule is naturally produced by the natural version of a bacterial cell (i.e. not engineered) provided herein but is produced by a different synthetic route and / or using different enzymes. In embodiments, the endogenously produced biomolecule is naturally produced by the natural version of a bacterial cell (i.e. not engineered) provided herein but is produced at lower levels than the bacterial cell provided herein.

[0122] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasingproliferation) or killing the cell. In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein (e.g. a cancer-associated protein). Similarly an "inhibitor" is a compound or protein that inhibits a receptor or another protein, e.g.,, by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0123] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0124] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0125] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population ofsimilar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier- obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, etc).

[0126] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0127] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.CELL COMPOSITIONS

[0128] In an aspect is provided a bacterial cell including a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein, wherein the bacterial cell does not express agyA gene product and agcvTHP gene product, and wherein the bacterial cell endogenously produces a 1 -carbon (Cl) metabolite. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl- THF cyclohydrolase (Fch) protein, and / or the methylene-THF dehydrogenase (MtdA) protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL)protein, the methenyl-THF cyclohydrolase (Fch) protein, and the methylene-THF dehydrogenase (MtdA) protein are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell.

[0129] In another aspect is provided a bacterial cell including a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product, and wherein the bacterial cell endogenously produces a formate. Where a bacterial cell provided herein endogenously produces formate, it is intended to mean that the formate is produced within the bacterial cell. As described herein, the endogenously produced formate may be made within the cell using exogenous enzymes as described herein.

[0130] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein. In embodiments, the bacterial cell includes a methenyl-THF cyclohydrolase (Fch) protein. In embodiments, the bacterial cell includes a methylene-THF dehydrogenase (MtdA) protein. the bacterial cell does not express ag / jM gene product and a gcvTHP gene product. In embodiments, the bacterial cell does not express a glyA gene product. In embodiments, the bacterial cell does not express a gcvTHP gene product.

[0132] In embodiments, all glyA genes and all gcvTHP genes are knocked out of the bacterial cell. In embodiments, all glyA genes are knocked out of the bacterial cell. In embodiments, eglyA-I gene is knocked out of the bacterial cell. In embodiments, the glyA - / I gene is knocked out of the bacterial cell. In embodiments, all gcvTHP genes are knocked out of the bacterial cell. In embodiments, the gcvT gene is knocked out of the bacterial cell. In embodiments, the gcvH gene is knocked out of the bacterial cell. In embodiments, the gcvP gene is knocked out of the bacterial cell. In embodiments, the gcvT gene, the gcvH gene, and the gcvP gene are knocked out of the bacterial cell. In embodiments, the Ipd gene is knocked out of the bacterial cell. In embodiments, the gcvT gene, the gcvH gene, the gcvP gene, and the Ipd gene are knocked out of the bacterial cell.

[0133] In embodiments, the bacterial cell endogenously produces a 1-carbon (Cl) metabolite. In embodiments, the Cl metabolite is a formate, a formaldehyde, a methanol, a methylamine, a methane, a carbon monoxide, a carbon dioxide, a carbonic acid, a dichloromethane, a methanethiol, or a methanesulfonate. In embodiments, the Cl metabolite is a formate. In embodiments, the Cl metabolite is a formaldehyde. In embodiments, the C l metabolite is a methanol. In embodiments, the Cl metabolite is a methylamine. In embodiments, the Cl metabolite is a methane. In embodiments, the Cl metabolite is a carbon monoxide. In embodiments, the Cl metabolite is a carbon dioxide. In embodiments, the Cl metabolite is a carbonic acid. In embodiments, the Cl metabolite is a dichloromethane. In embodiments, the Cl metabolite is a methanethiol. In embodiments, the Cl metabolite is a methanesulfonate. Where a bacterial cell provided herein endogenously produces a Cl metabolite, it is intended to mean that the Cl metabolite is produced within the bacterial cell. As described herein, the endogenously produced Cl metabolite may be made within the cell using exogenous enzymes as described herein.

[0134] In embodiments, the bacterial cell further includes a formamidase protein, a cyanide dihydratase protein, a formate dehydrogenase protein, or a formaldehyde dehydrogenase protein. In embodiments, the bacterial cell further includes a formamidase protein. In embodiments, the bacterial cell further includes a cyanide dihydratase protein. In embodiments, the bacterial cell further includes a formate dehydrogenase protein. In embodiments, the bacterial cell further includes a formaldehyde dehydrogenase protein. In embodiments, one or more of the formamidase protein, the cyanide dihydratase protein, the formate dehydrogenase protein, and / or the formaldehyde dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the formamidase protein, the cyanide dihydratase protein, the formate dehydrogenase protein, and the formaldehyde dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the formamidase protein is exogenous to the bacterial cell. In embodiments, the cyanide dihydratase protein is exogenous to the bacterial cell. In embodiments, the formate dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell.

[0135] In embodiments, the bacterial cell further includes a cyanide hydratase protein, a carbon monoxide dehydrogenase protein, a carbonic anhydrase protein, a dichloromethane dehalogenase protein, a methanethiol oxidase protein, a methanesulfonate monooxygenase protein, a methanoldehydrogenase protein, or a methylamine dehydrogenase protein. In embodiments, the bacterial cell further includes a cyanide hydratase protein. In embodiments, the bacterial cell further includes a carbon monoxide dehydrogenase protein. In embodiments, the bacterial cell further includes a carbonic anhydrase protein. In embodiments, the bacterial cell further includes a dichloromethane dehalogenase protein. In embodiments, the bacterial cell further includes a methanethiol oxidase protein. In embodiments, the bacterial cell further includes a methanesulfonate monooxygenase protein. In embodiments, the bacterial cell further includes a methanol dehydrogenase protein. In embodiments, the bacterial cell further includes a methylamine dehydrogenase protein. In embodiments, one or more of the cyanide hydratase protein, the carbon monoxide dehydrogenase protein, the carbonic anhydrase protein, the dichloromethane dehalogenase protein, the methanethiol oxidase protein, the methanesulfonate monooxygenase protein, the methanol dehydrogenase protein, and / or the methylamine dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the cyanide hydratase protein, the carbon monoxide dehydrogenase protein, the carbonic anhydrase protein, the dichloromethane dehalogenase protein, the methanethiol oxidase protein, the methanesulfonate monooxygenase protein, the methanol dehydrogenase protein, and the methylamine dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the cyanide hydratase protein is exogenous to the bacterial cell. In embodiments, the carbon monoxide dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the carbonic anhydrase protein is exogenous to the bacterial cell. In embodiments, the dichloromethane dehalogenase protein is exogenous to the bacterial cell. In embodiments, the methanethiol oxidase protein is exogenous to the bacterial cell. In embodiments, the methanesulfonate monooxygenase protein is exogenous to the bacterial cell. In embodiments, the methanol dehydrogenase protein is exogenous to the bacterial cell. In embodiments, and the methylamine dehydrogenase protein is exogenous to the bacterial cell.

[0136] In embodiments, the bacterial cell further includes a methane monooxygenase protein. In embodiments, the methane monooxygenase protein is exogenous to the bacterial cell.

[0137] In embodiments, the bacterial cell further includes a tryptophan dioxygenase (TDO) protein and a kynurenine formamidase (KF A) protein. In embodiments, the bacterial cell produces an L-3-hydroxykynurenine. In embodiments, one or more of the tryptophan dioxygenase (TDO)protein and / or the kynurenine formamidase (KF A) protein is / are exogenous to the bacterial cell. In embodiments, the tryptophan dioxygenase (TDO) protein and the kynurenine formamidase (KF A) protein are exogenous to the bacterial cell. In embodiments, the tryptophan dioxygenase (TDO) protein is exogenous to the bacterial cell. In embodiments, the kynurenine formamidase (KF A) protein is exogenous to the bacterial cell.

[0138] In embodiments, the bacterial cell further includes a kynurenine monooxygenase (KMO) protein. In embodiments, the kynurenine monooxygenase (KMO) protein is exogenous to the bacterial cell.

[0139] In embodiments, the bacterial cell includes a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and a kynurenine monooxygenase (KMO) protein. In embodiments, the bacterial cell includes a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, or a kynurenine monooxygenase (KMO) protein. In embodiments, the bacterial cell includes a tryptophan dioxygenase (TDO) protein. In embodiments, the bacterial cell includes a kynurenine formamidase (KF A) protein. In embodiments, the bacterial cell includes a kynurenine monooxygenase (KMO) protein. In embodiments, one or more of the tryptophan dioxygenase (TDO) protein, the kynurenine formamidase (KF A) protein, and / or the kynurenine monooxygenase (KMO) protein is / are exogenous to the bacterial cell. In embodiments, the tryptophan dioxygenase (TDO) protein, the kynurenine formamidase (KF A) protein, and the kynurenine monooxygenase (KMO) protein are exogenous to the bacterial cell. In embodiments, the tryptophan dioxygenase (TDO) protein is exogenous to the bacterial cell. In embodiments, the kynurenine formamidase (KF A) protein is exogenous to the bacterial cell. In embodiments, the kynurenine monooxygenase (KMO) protein is exogenous to the bacterial cell.

[0140] In embodiments, the bacterial cell produces a xanthommatin (Xa).

[0141] In embodiments, the bacterial cell further includes a kynurenine aminotransferase protein, a kynureninase protein, or an aromatic L-amino acid decarboxylase protein. In embodiments, the bacterial cell further includes a kynurenine aminotransferase protein. In embodiments, the bacterial cell further includes a kynureninase protein. In embodiments, the bacterial cell further includes an aromatic L-amino acid decarboxylase. In embodiments, one or more of the kynurenineaminotransferase protein, the kynureninase protein, and / or the aromatic L-amino acid decarboxylase protein is / are exogenous to the bacterial cell. In embodiments, the kynurenine aminotransferase protein, the kynureninase protein, and the aromatic L-amino acid decarboxylase protein are exogenous to the bacterial cell. In embodiments, the kynurenine aminotransferase protein is exogenous to the bacterial cell. In embodiments, the kynureninase protein is exogenous to the bacterial cell. In embodiments, the aromatic L-amino acid decarboxylase protein is exogenous to the bacterial cell.

[0142] In embodiments, the bacterial cell produces a kynurenic acid, an anthranilic acid, a kynuramine, a xanthurenic acid, a 3-hydroxyanthranilic acid, a cinnabarinic acid, or a 3- hydroxykynuramine. In embodiments, the bacterial cell produces a kynurenic acid. In embodiments, the bacterial cell produces an anthranilic acid. In embodiments, the bacterial cell produces a kynuramine. In embodiments, the bacterial cell produces a xanthurenic acid. In embodiments, the bacterial cell produces a 3-hydroxyanthranilic acid. In embodiments, the bacterial cell produces a cinnabarinic acid. In embodiments, the bacterial cell produces a 3-hydroxykynuramine.

[0143] In embodiments, the bacterial cell further includes a 3-hydroxyanthranilic acid oxidase protein or a 3-hydroxyanthranilic acid oxygenase protein. In embodiments, the bacterial cell further includes a 3-hydroxyanthranilic acid oxidase protein. In embodiments, the bacterial cell further includes a 3-hydroxyanthranilic acid oxygenase protein. In embodiments, one or more of the 3- hydroxyanthranilic acid oxidase protein and / or the 3-hydroxyanthranilic acid oxygenase protein is / are exogenous to the bacterial cell. In embodiments, the 3-hydroxyanthranilic acid oxidase protein and the 3-hydroxyanthranilic acid oxygenase protein are exogenous to the bacterial cell. In embodiments, the 3-hydroxyanthranilic acid oxidase protein is exogenous to the bacterial cell. In embodiments, the 3-hydroxyanthranilic acid oxygenase protein is exogenous to the bacterial cell.

[0144] In embodiments, the bacterial cell produces a quinolinic acid or a picolinic acid. In embodiments, the bacterial cell produces a quinolinic acid. In embodiments, the bacterial cell produces a picolinic acid.

[0145] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase(MtdA) protein, and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, and / or the methylene-THF dehydrogenase (MtdA) protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, and the methylene-THF dehydrogenase (MtdA) protein are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell.

[0146] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, and a formamidase protein, and the bacterial cell does not express a glyA gene product and a gcvTHP gene product In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, and / or the formamidase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, and the formamidase protein are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formamidase protein is exogenous to the bacterial cell.

[0147] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, and a formate dehydrogenase protein, and the bacterial cell does not express a glyA gene product and a gcvTHP gene product In embodiments, one or more of the formate- tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, and / or the formate dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL)protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, and the formate dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the format e-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formate dehydrogenase protein is exogenous to the bacterial cell.

[0148] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formate dehydrogenase protein, and a carbon monoxide dehydrogenase protein; and the bacterial cell does not express a / jM gene product and a gcvTHP gene product In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formate dehydrogenase protein, and / or the carbon monoxide dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formate dehydrogenase protein, and the carbon monoxide dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formate dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the carbon monoxide dehydrogenase protein is exogenous to the bacterial cell.

[0149] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formate dehydrogenase protein, and a carbonic anhydrase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formate dehydrogenase protein, and / or the carbonic anhydrase protein is / are exogenous to the bacterial cell. In embodiments, theformate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formate dehydrogenase protein, and the carbonic anhydrase protein are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formate dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the carbonic anhydrase protein is exogenous to the bacterial cell.

[0150] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formamidase protein, and a cyanide hydratase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formamidase protein, and / or the cyanide hydratase protein is / are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formamidase protein, and the cyanide hydratase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formamidase protein is exogenous to the bacterial cell. In embodiments, the cyanide hydratase protein is exogenous to the bacterial cell.

[0151] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, and a cyanide dihydratase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, one or more of the formate- tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formamidase protein, and / or the cyanidedihydratase protein is / are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formamidase protein, and the cyanide dihydratase protein are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formamidase protein is exogenous to the bacterial cell. In embodiments, the cyanide dihydratase protein is exogenous to the bacterial cell.

[0152] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, and a formaldehyde dehydrogenase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, one or more of the formate- tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, and / or the formaldehyde dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, and the formaldehyde dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formamidase protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell.

[0153] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formaldehyde dehydrogenase protein, and a methylamine dehydrogenase protein; and the bacterial cell does not express a / jM gene product and a gcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehydedehydrogenase protein, and / or the methylamine dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formamidase protein, and the methylamine dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the methylamine dehydrogenase protein is exogenous to the bacterial cell.

[0154] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formaldehyde dehydrogenase protein, and a dichloromethane dehalogenase protein; and the bacterial cell does not express aglyA gene product and agcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl- THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and / or the dichloromethane dehalogenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and the dichloromethane dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the dichloromethane dehalogenase protein is exogenous to the bacterial cell.

[0155] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formaldehyde dehydrogenase protein, and a methanethiol oxidase protein; and thebacterial cell does not express agfyA gene product and gcvTHP gene product In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and / or the methanethiol oxidase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and the methanethiol oxidase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the methanethiol oxidase protein is exogenous to the bacterial cell.

[0156] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formaldehyde dehydrogenase protein, and a methanesulfonate monooxygenase protein; and the bacterial cell does not express glyA gene product and gcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl- THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and / or the methanesulfonate monooxygenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and the methanesulfonate monooxygenase protein are exogenous to the bacterial cell. In embodiments, the formate- tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the methanesulfonate monooxygenase protein is exogenous to the bacterial cell.

[0157] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formaldehyde dehydrogenase protein, and a methanol dehydrogenase protein; and the bacterial cell does not express agfyA gene product and a gcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and / or the methanol dehydrogenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, and the methanol dehydrogenase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch) protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the methanol dehydrogenase protein is exogenous to the bacterial cell.

[0158] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a formaldehyde dehydrogenase protein, a methanol dehydrogenase protein, and a methane monooxygenase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, one or more of the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, the methanol dehydrogenase protein, and / or the methane monooxygenase protein is / are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein, the methenyl-THF cyclohydrolase (Fch) protein, the methylene-THF dehydrogenase (MtdA) protein, the formaldehyde dehydrogenase protein, the methanol dehydrogenase protein, and the methane monooxygenase protein are exogenous to the bacterial cell. In embodiments, the formate-tetrahydrofolate(THF) ligase (FtfL) protein is exogenous to the bacterial cell. In embodiments, the methenyl-THF cyclohydrolase (Fch)protein is exogenous to the bacterial cell. In embodiments, the methylene-THF dehydrogenase (MtdA) protein is exogenous to the bacterial cell. In embodiments, the formaldehyde dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the methanol dehydrogenase protein is exogenous to the bacterial cell. In embodiments, the methane monooxygenase protein is exogenous to the bacterial cell.

[0159] In some aspects provided herein, a bacterial cell provided herein including embodiments thereof may be used in a method of producing a biomolecule. In embodiments, the method includes culturing a bacterial cell provided herein including embodiments thereof in an appropriate cell growth media.

[0160] In embodiments, the cell growth media includes an exogenous C 1 metabolite in the cell growth media. In embodiments, the cell growth media includes an exogenous Cl metabolite in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous Cl metabolite in the cell growth media. In embodiments, the cell growth media includes an exogenous formate in the cell growth media. In embodiments, the cell growth media includes an exogenous formate in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous formate in the cell growth media. In embodiments, the cell growth media includes an exogenous carbon dioxide in the cell growth media. In embodiments, the cell growth media includes an exogenous carbon dioxide in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous carbon dioxide in the cell growth media. In embodiments, the cell growth media includes an exogenous carbonic acid in the cell growth media. In embodiments, the cell growth media includes an exogenous carbonic acid in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous carbonic acid in the cell growth media. In embodiments, the cell growth media includes an exogenous carbon monoxide in the cell growth media. In embodiments, the cell growth media includes an exogenous carbon monoxide in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous carbon monoxide in the cell growth media. In embodiments, the cell growth media includes an exogenous hydrogen cyanide in the cell growth media. In embodiments, the cell growth media includes an exogenoushydrogen cyanide in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous hydrogen cyanide in the cell growth media. In embodiments, the cell growth media includes an exogenous formamide in the cell growth media. In embodiments, the cell growth media includes an exogenous formamide in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous formamide in the cell growth media. In embodiments, the cell growth media includes an exogenous formaldehyde in the cell growth media. In embodiments, the cell growth media includes an exogenous formaldehyde in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous formaldehyde in the cell growth media. In embodiments, the cell growth media includes an exogenous dichloromethane in the cell growth media. In embodiments, the cell growth media includes an exogenous di chloromethane in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous dichloromethane in the cell growth media. In embodiments, the cell growth media includes an exogenous methanethiol in the cell growth media. In embodiments, the cell growth media includes an exogenous methanethiol in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous methanethiol in the cell growth media. In embodiments, the cell growth media includes an exogenous methanesulfonate in the cell growth media. In embodiments, the cell growth media includes an exogenous methanesulfonate in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous methanesulfonate in the cell growth media. In embodiments, the cell growth media includes an exogenous methanol in the cell growth media. In embodiments, the cell growth media includes an exogenous methanol in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous methanol in the cell growth media. In embodiments, the cell growth media includes an exogenous methylamine in the cell growth media. In embodiments, the cell growth media includes an exogenous methylamine in the cell growth media in an insufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous methylamine in the cell growth media. In embodiments, the cell growth media includes an exogenous methane in the cell growth media. In embodiments, the cell growth media includes an exogenous methane in the cell growth media in aninsufficient amount for cell growth. In embodiments, the cell growth media does not include an exogenous methane in the cell growth media.

[0161] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, and a tryptophan dioxygenase (TDO) protein; and the bacterial cell does not express a glyA gene product and agcvTHP gene product. In embodiments, the bacterial cell produces an N- formylkynurenine. Where a bacterial cell provided herein endogenously produces N- formylkynurenine, it is intended to mean that the N-formylkynurenine is produced within the bacterial cell. As described herein, the endogenously produced N-formylkynurenine may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce N-formylkynurenine. In embodiments, the method of producing N- formylkynurenine includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0162] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, and a kynurenine formamidase (KF A) protein; and the bacterial cell does not express a glyA gene product and agcvTHP gene product. In embodiments, the bacterial cell produces an L-kynurenine. Where a bacterial cell provided herein endogenously produces L-kynurenine, it is intended to mean that the L-kynurenine is produced within the bacterial cell. As described herein, the endogenously produced L-kynurenine may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce L-kynurenine. In embodiments, the method of producing L- kynurenine includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0163] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and a kynurenine aminotransferase protein; and the bacterial cell does not express a glyA gene product and agcvTHP gene product. In embodiments, the bacterial cell produces a kynurenic acid. Where abacterial cell provided herein endogenously produces kynurenic acid, it is intended to mean that the kynurenic acid is produced within the bacterial cell. As described herein, the endogenously produced kynurenic acid may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce kynurenic acid. In embodiments, the method of producing kynurenic acid includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0164] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and a kynureninase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, the bacterial cell produces an anthranilic acid. Where a bacterial cell provided herein endogenously produces anthranilic acid, it is intended to mean that the anthranilic acid is produced within the bacterial cell. As described herein, the endogenously produced anthranilic acid may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce anthranilic acid. In embodiments, the method of producing anthranilic acid includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0165] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and an aromatic L-amino acid decarboxylase protein; and the bacterial cell does not express a glyA gene product and agcvlH' P gene product. In embodiments, the bacterial cell produces a kynuramine. Where a bacterial cell provided herein endogenously produces kynuramine, it is intended to mean that the kynuramine is produced within the bacterial cell. As described herein, the endogenously produced kynuramine may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce kynuramine. In embodiments, the method of producing kynuramine includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0166] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and a kynurenine monooxygenase (KMO) protein; and the bacterial cell does not express a glyA gene product and agcvTHP gene product. In embodiments, the bacterial cell produces a L-3- hydroxykynurenine, a xanthommatin (Xa), or a decarboxylated xanthommatin. In embodiments, the bacterial cell produces a L-3-hydroxykynurenine. In embodiments, the bacterial cell produces a xanthommatin (Xa). In embodiments, the bacterial cell produces a decarboxylated xanthommatin. Where a bacterial cell provided herein endogenously produces xanthommatin, it is intended to mean that the xanthommatin is produced within the bacterial cell. As described herein, the endogenously produced xanthommatin may be made within the cell using exogenous enzymes as described herein. Where a bacterial cell provided herein endogenously produces decarboxylated xanthommatin, it is intended to mean that the decarboxylated xanthommatin is produced within the bacterial cell. In embodiments, the bacterial cell may be used in a method to produce xanthommatin. In embodiments, the method of producing xanthommatin includes culturing the bacterial cell provided herein in an appropriate cell growth media. As described herein, the endogenously produced decarboxylated xanthommatin may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce decarboxylated xanthommatin. In embodiments, the method of producing decarboxylated xanthommatin includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0167] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, a kynurenine monooxygenase (KMO) protein, and a kynurenine aminotransferase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, the bacterial cell produces a xanthurenic acid. Where a bacterial cell provided herein endogenously produces xanthurenic acid, it is intended to mean that the xanthurenic acid is produced within the bacterial cell. As described herein, the endogenously produced xanthurenic acid may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may beused in a method to produce xanthurenic acid. In embodiments, the method of producing xanthurenic acid includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0168] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, a kynurenine monooxygenase (KMO) protein, and an aromatic L-amino acid decarboxylase protein; and the bacterial cell does not express aglyA gene product and gcvTHP gene product. In embodiments, the bacterial cell produces a 3-hydroxykynuramine. Where a bacterial cell provided herein endogenously produces 3-hydroxykynuramine, it is intended to mean that the 3- hydroxykynuramine is produced within the bacterial cell. As described herein, the endogenously produced 3-hydroxykynuramine may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce 3- hydroxykynuramine. In embodiments, the method of producing 3-hydroxykynuramine includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0169] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, a kynurenine monooxygenase (KMO) protein, and a kynureninase protein; and the bacterial cell does not express ag / yA gene product and a gcvTHP gene product. In embodiments, the bacterial cell produces a 3-hydroxyanthranilic acid or cinnabarinic acid. In embodiments, the bacterial cell produces a 3-hydroxyanthranilic acid. In embodiments, the bacterial cell produces cinnabarinic acid. Where a bacterial cell provided herein endogenously produces cinnabarinic acid, it is intended to mean that the cinnabarinic acid is produced within the bacterial cell. As described herein, the endogenously produced cinnabarinic acid may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce cinnabarinic acid. In embodiments, the method of producing cinnabarinic acid includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0170] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, a kynurenine monooxygenase (KMO) protein, a kynureninase protein, and a 3-hydroxyanthranilic acid oxygenase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, the bacterial cell produces a picolinic acid. Where a bacterial cell provided herein endogenously produces picolinic acid, it is intended to mean that the picolinic acid is produced within the bacterial cell. As described herein, the endogenously produced picolinic acid may be made within the cell using exogenous enzymes as described herein. In embodiments, the bacterial cell may be used in a method to produce picolinic acid. In embodiments, the method of producing picolinic acid includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0171] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, a methylene-THF dehydrogenase (MtdA) protein, a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, a kynurenine monooxygenase (KMO) protein, a kynureninase protein, and a 3-hydroxyanthranilic acid oxidase protein; and the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, the bacterial cell produces a quinolinic acid. Where a bacterial cell provided herein endogenously produces quinolinic acid, it is intended to mean that the quinolinic acid is produced within the bacterial cell. As described herein, the endogenously produced quinolinic acid may be made within the cell using exogenous enzymes as described herein. . In embodiments, the bacterial cell may be used in a method to produce quinolinic acid. In embodiments, the method of producing quinolinic acid includes culturing the bacterial cell provided herein in an appropriate cell growth media.

[0172] In embodiments, the bacterial cell is a Pseudomonadota bacterial cell. In embodiments, the bacterial cell is an Acidithiobacillia bacterial cell, an Alphaproteobacteria bacterial cell, a Betaproteobacteria bacterial cell, a Gammaproteobacteria bacterial cell, a Hydrogenophilia bacterial cell, or a Zetaproteobacteria bacterial cell. In embodiments, the bacterial cell is an Acidithiobacillia bacterial cell. In embodiments, the bacterial cell is an Alphaproteobacteria bacterial cell. Inembodiments, the bacterial cell is a Betaproteobacteria bacterial cell. In embodiments, the bacterial cell is a Gammaproteobacteria bacterial cell. In embodiments, the bacterial cell is a Hydrogenophili a bacterial cell. In embodiments, the bacterial cell is or a Zetaproteobacteria bacterial cell.

[0173] In embodiments, the bacterial cell is a Pseudomonas bacterial cell. In embodiments, the bacterial cell is a Pseudomonas putida cell. In embodiments, the Pseudomonas putida cell is a Pseudomonas putida KT2440 cell.

[0174] In embodiments, the bacterial cell is a Bacillus bacterial cell. In embodiments, the Bacillus bacterial cell is a Bacillus subtilis bacterial cell.

[0175] In embodiments, the bacterial cell is a Actinomycetota bacterial cell. In embodiments, the Actinomycetota bacterial cell is a Streptomyces bacterial cell. In embodiments, the Streptomyces bacterial cell is a Streptomyces coelicolor bacterial cell.

[0176] In another aspect is provided a bacterial cell including a xanthommatin (Xa).

[0177] In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein. In embodiments, the bacterial cell includes a formate-tetrahydrofolate(THF) ligase (FtfL) protein. In embodiments, the bacterial cell includes a methenyl-THF cyclohydrolase (Fch) protein. In embodiments, the bacterial cell includes a methylene-THF dehydrogenase (MtdA) protein.

[0178] In embodiments, the bacterial cell does not express a glyA gene product and a gcvTHP gene product. In embodiments, the bacterial cell does not express a glyA gene product. In embodiments, the bacterial cell does not express a gcvTHP gene product.

[0179] In embodiments, all glyA genes and all gcvTHP genes are knocked out of the bacterial cell. In embodiments, all glyA genes are knocked out of the bacterial cell. In embodiments, the.glyA-1 gene is knocked out of the bacterial cell. In embodiments, the glyA-II gene is knocked out of the bacterial cell. In embodiments, all gcvTHP genes are knocked out of the bacterial cell. In embodiments, the gcvT gene is knocked out of the bacterial cell. In embodiments, the gcvH gene is knocked out of the bacterial cell. In embodiments, the gcvP gene is knocked out of the bacterial cell.In embodiments, the gcvT gene, the gcvH gene, and the gcvP gene are knocked out of the bacterial cell. In embodiments, the Ipd gene is knocked out of the bacterial cell. In embodiments, the gcvT gene, the gcvH gene, the gcvP gene, and the Ipd gene are knocked out of the bacterial cell.

[0180] In embodiments, the bacterial cell includes a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and a kynurenine monooxygenase (KMO) protein. In embodiments, the bacterial cell includes a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, or a kynurenine monooxygenase (KMO) protein. In embodiments, the bacterial cell includes a tryptophan dioxygenase (TDO) protein. In embodiments, the bacterial cell includes a kynurenine formamidase (KF A) protein. In embodiments, the bacterial cell includes a kynurenine monooxygenase (KMO) protein.

[0181] In embodiments, the bacterial cell is applied to a surface or a hydrogel. In embodiments, the bacterial cell is applied to a surface. In embodiments, the bacterial cell is applied to a hydrogel.

[0182] In another aspect is provided a cell, wherein the cell includes an auxotroph. In embodiments, the auxotroph includes a 1-carbon (Cl) metabolite auxotroph. In embodiments, the cell is unable to synthesize a cofactor in the absence of the Cl metabolite. In embodiments, the cell is capable of synthesizing a biomolecule.

[0183] In embodiments, the cell does not include ag / jM gene and / or a gcvTHP gene. In embodiments, the cell does not include a / jM gene and a gcvTHP gene. In embodiments, the cell does not include a glyA gene or a gcvTHP gene. In embodiments, the cell does not include a glyA gene. In embodiments, the cell does not include a gcvTHP gene. In embodiments, th e g / yd gene encodes a serine hydroxymethyltransferase protein. In embodiments, the gcvTHP gene encodes enzymes of the glycine cleavage system.

[0184] In embodiments, the cell includes a formate-THF ligase protein (FtfL), a methenyl-THF cyclohydrolase protein (Fch), and / or a methylene-THF dehydrogenase protein (MtdA). In embodiments, the cell includes a formate-THF ligase protein (FtfL), a methenyl-THF cyclohydrolase protein (Fch), and a methylene-THF dehydrogenase protein (MtdA). In embodiments, the cell includes a formate-THF ligase protein (FtfL), a methenyl-THF cyclohydrolase protein (Fch), or a methylene-THF dehydrogenase protein (MtdA). In embodiments,the cell includes a formate-THF ligase protein (FtfL). In embodiments, the cell includes a methenyl- THF cyclohydrolase (Fch). In embodiments, the cell includes a methylene-THF dehydrogenase protein (MtdA).

[0185] In embodiments, the cofactor includes 5,10-methylene-THF (MTHF). In embodiments, the Cl metabolite includes a formate, a formaldehyde, a methanol, a methylamine, a methane, a carbon monoxide, and / or a carbon dioxide. In embodiments, the C 1 metabolite includes a formate, a formaldehyde, a methanol, a methylamine, a methane, a carbon monoxide, and a carbon dioxide. In embodiments, the Cl metabolite includes a formate, a formaldehyde, a methanol, a methylamine, a methane, a carbon monoxide, or a carbon dioxide. In embodiments, the Cl metabolite includes a formate, a formaldehyde. In embodiments, the Cl metabolite includes a methanol. In embodiments, the Cl metabolite includes a methylamine. In embodiments, the Cl metabolite includes a methane. In embodiments, the Cl metabolite includes a carbon monoxide. In embodiments, the Cl metabolite includes a carbon dioxide.

[0186] In embodiments, the auxotroph further includes a glycine auxotroph. In embodiments, the cell does not include a ItaE gene. In embodiments, the ItaE gene encodes a L-threonine aldolase protein.

[0187] In embodiments, the cell further includes a tryptophan dioxygenase protein (TDO), a kynurenine formamidase protein (KF A), and / or a kynurenine monooxygenase protein (KMO). In embodiments, the cell further includes a tryptophan dioxygenase protein (TDO), a kynurenine formamidase protein (KF A), and a kynurenine monooxygenase protein (KMO). In embodiments, the cell further includes a tryptophan dioxygenase protein (TDO), a kynurenine formamidase protein (KF A), or a kynurenine monooxygenase protein (KMO). In embodiments, the cell further includes a tryptophan dioxygenase protein (TDO). In embodiments, the cell further includes a kynurenine formamidase protein (KF A). In embodiments, the cell further includes a kynurenine monooxygenase protein (KMO).

[0188] In embodiments, the cell synthesizes a 3-hydroxykynurenine (3-HK). In embodiments, the 3-HK is dimerized by oxidative cyclization. In embodiments, the oxidative cyclization includes a heme-peroxidase. In embodiments, the biomolecule includes a xanthommatin (Xa).

[0189] In embodiments, wherein the cell includes a bacterial cell. In embodiments, the bacterial cell includes Pseudomonas putida cell. In embodiments, the Pseudomonas putida cell includes a Pseudomonas putida KT2440 cellMETHODS

[0190] In an aspect is provided a method for synthesizing a biomolecule, the method including: (a) culturing the cell provided herein including embodiments thereof; and (b) allowing the cell to synthesize the biomolecule. In embodiments, the synthesis of the biomolecule is coupled to cell growth and / or survival.

[0191] In embodiments, the biomolecule includes 5,10-methylene-THF (MTHF), 3- hydroxy kynurenine (3-HK), xanthommatin (Xa), and methyl anthranilate. In embodiments, the biomolecule includes 5,10-methylene-THF (MTHF), 3 -hydroxy kynurenine (3-HK), xanthommatin (Xa), or methyl anthranilate. In embodiments, the biomolecule includes 5,10-methylene-THF (MTHF), 3 -hydroxy kynurenine (3-HK), xanthommatin (Xa), and / or methyl anthranilate. In embodiments, the biomolecule includes 5,10-methylene-THF (MTHF). In embodiments, the biomolecule includes 3 -hydroxy kynurenine (3-HK). In embodiments, the biomolecule includes xanthommatin (Xa). In embodiments, the biomolecule includes methyl anthranilate.EXAMPLESExample 1

[0192] In embodiments, the engineered organism with endogenously produced formate functions under selective conditions. The selective media is supplemented with formate (exogenous) or a heterologous enzyme / pathway is incorporated into the strain that generates formate from the relevant substrate. In this case, enzyme / pathway activity rescues auxotrophy by generating formate. This results in growth-coupling. The growth-coupling strategy can be used for adaptive laboratory evolution or as a selection in directed evolution.

[0193] A Pseudomonas putida C 1 auxotrophic strain harbors a genetic pathway that encodes the biosynthetic enzymes for production of the target compounds. When the strain is grown under selective conditions, the sole source of formate is supplied by this pathway. In this way, production of the target compound provides a survival advantage that drives flux through the pathway.

[0194] The strain has the potential to be applied as a chassis for bacterial cell factories that release Cl -products en route to the target molecule. The strain can also be used as a selection strategy for directed evolution of enzymes / pathways that generate Cl -products, such as demethylases and decarboxylases.

[0195] In embodiments, the cells provided herein may be used for biobased production of L- kynurenine, L-3 -hydroxykynurenine, and xanthommatin pigments. Xanthommatin is being tested as a biomaterial in paint, cosmetics, sensors, etc. in academia, industry, and government. Presently it is made synthetically from two molecules of L-3-hydroxykynurenine, which is a rare and expensive metabolite.Example 1: General Methods and Materials

[0196] Cl / Formate Auxotroph, We engineered Pseudomonas putida KT2440 formate auxotroph. In native P. putida metabolism, biosynthesis of the essential cofactor 5,10-methylene-THF (MTHF) is supported by the activities of serine hydroxymethyltransferase (encoded by fyA)' and the enzymes of the glycine cleavage system (encoded by gcvTHP). In our design, genes glyA and gcvTHP are deleted to abolish endogenous biosynthesis of MTHF. Instead, MTHF is derived from direct ligation of formate to THF. This is enabled by introduction of a formate assimilation pathway from Methylobacterium extorquens, composed of formate-THF ligase (FtfL), methenyl-THF cyclohydrolase (Fch), and methylene-THF dehydrogenase (MtdA). The result is a formate-requiring auxotrophic strain, that under selective conditions, only grows if supplied with exogenous or endogenously produced formate.

[0197] Growth is also rescued by other exogenous or endogenously produced Cl -compounds, including formaldehyde and methanol due to activity of native enzymes formaldehyde dehydrogenase and methanol dehydrogenase, respectively. Strain derivatives can also be engineered for other Cl -compounds, such as methylamine, methane, carbon monoxide, and carbon dioxide by incorporating appropriate pathways for conversion to formate.

[0198] Glycine auxotroph derivative. Gene ItaE, which encodes L-threonine aldolase, is deleted to abolish biosynthesis of glycine from threonine. This derivative strain requires both glycine and formate for growth.

[0199] Growth-coupled biosynthesis. Pathways / enzymes that release Cl -byproducts function as a source of formate, linking them to biosynthesis of the essential cofactor MTHF, and thus to growth. The organism is forced to direct metabolic flux towards production of the target compound to survive. Our system can thus be applied to:1) Bioproduction of molecules that are biosynthesized via Cl-releasing enzymes;2) Growth-coupled adaptive laboratory evolution; and3) Growth selection screens such as for directed evolution of Cl-releasing enzymes / pathway s .Example 3: Bioproduction of animal pigment xanthommatin

[0200] As a proof-of-concept, we applied the Cl -based growth-coupling strategy to bioproduction of L-3 -hydroxy kynurenine (3-HK) L-3 -hydroxy kynurenine (3-HK) is biosynthesized by a three- enzyme pathway, consisting of tryptophan dioxygenase (TDO), kynurenine formamidase (KF A), and kynurenine monooxygenase (KMO). In the first step, L-Trp is oxidized to N-formyl-kynurenine by heme dependent 2,3- tryptophan dioxygenase (TDO) Next, the formyl group is removed by yielding kynurenine (Kyn) and releasing formate. Flavin-dependent kynurenine monooxygenase (KMO) then catalyzes hydroxylation of Kyn to 3-HK. A three gene biosynthetic pathway, composed of TDO, KFA and KMO, were constructed and transformed into the auxotrophic strain. Both pathways rescued growth and produced biosynthetic intermediates, side products, and 3-HK at higher titers relative to wildtype P. putida KT2440 carrying identical pathways. The 3-HK producing Cl-auxotroph also produced the pigment xanthommatin (Xa), which is the result of dimerization of 3-HK followed by oxidative cyclization. Promiscuous activities of native heme- peroxidases are likely catalyzing formation of Xa from 3-HK. A derivative strain, that lacks KMO, was also generated, for overproduction of L-kynurenine. This strain can function as a platform strain for bioproduction of other kynurenine-derived products, such as the flavoring ingredient methyl anthranilate. We observed the strain produced all three metabolites, including Xa at ~0.4 mg / mL.Example 4

[0201] We designed a bacterial strain to couple growth with the targeted production of a biomolecule. This is a new strategy of growth-coupled biosynthesis in which a byproduct of themetabolic production of the desired target molecule is absolutely required to cure a metabolic deficiency of the engineered bacterial host chassis. In other words, if the host does not produce the designed product, then the host will not grow. We thus designed and built an engineered bacterial strain and showcased the microbial production of a high-value animal biopigment. In embodiments, there are two notable engineering elements to the cells provided herein.

[0202] First, we genetically engineered the bacterium Pseudomonas putida KT2440 to become an auxotroph for 1-carbon (Cl) metabolites such as formate, formaldehyde, or methanol. The foundational strain that we engineered is unable to synthesize the essential cofactor 5,10-methylene- THF (MTHF) in the absence of exogenous or endogenously produced formate. We blocked native MTHF biosynthesis from serine and glycine via deletions in serine hydroxymethyltransferase (glyA) and the glycine cleavage system (gcvTHP). We incorporated a synthetic operon consisting of formate-tetrahydrofolate ligase, 5,10-methenyltetrahydrofolate cyclohydrolase, and 5,10-methylene tetrahydrofolate dehydrogenase into the genome to allow for MTHF biosynthesis from formate. Under selective conditions, the strain can only survive when supplied with formate. This strain is useful for growth-coupling enzymes / pathways that generate formate. The growth-coupling strategy can be used for adaptive laboratory evolution or as a selection in directed evolution. We engineered additional strains to instead utilize formaldehyde or methanol which are converted into formate via additional gene insertions.

[0203] Second, we engineered the C l Pseudomonas putida auxotroph strain to biosynthesize the cephalopod pigment xanthommatin (Xa) and survive via one-carbon metabolism. The formatedependent Cl -auxotroph was engineered to harbor the biosynthetic enzymes for production of the tryptophan-derived metabolites L-kynurenine, L-3-hydroxykynurenine, and xanthommatin. In the course of the 4-step biosynthesis, formate is generated as a by-product during the second metabolic reaction via the enzyme kynurenine formamidase, resulting in a growth-coupled system in which production of these metabolites is directly tied to survival. Growth-coupling drives flux through the biosynthetic pathway resulting in >100x higher titer as compared to wildtype. The biomanufacturing of Xa addresses a mounting need in materials science to biosynthetically produce Xa due to its promise and potential as a high-refractive index, electrochromic molecule with the capability of templating camouflaging materials.References

[0204] (1) Turlin, J.; Dronsella, B.; Maria, A. D.; Lindner, S. N.; Nikel, P. I. Integrated Rational and Evolutionary Engineering of Genome-Reduced Pseudomonas putida Strains Empowers Synthetic Formate Assimilation. bioRxiv 2022, 2022.07.10.499488. doi.org / 10, 1101 / 2022,07.10,499488.

[0205] (2) Yishai, O.; Goldbach, L.; Tenenboim, H.; Lindner, S. N.; Bar-Even, A. Engineered Assimilation of Exogenous and Endogenous Formate in Escherichia coli. ACS Synth.Biol. 2017, 6 (9), 1722-1731. doi.org / 10.1021 / acssynbio.7b00Q86.

[0206] (3) Claassens, N. J.; Bordanaba-Florit, G.; Cotton, C. A. R.; De Maria, A.; Finger-Bou, M.; Friedeheim, L.; Giner-Laguarda, N.; Munar-Palmer, M.; Newell, W .; Scarinci, G.; Verbunt, J.; de Vries, S. T.; Yilmaz, S.; Bar-Even, A. Replacing the Calvin Cycle with the Reductive Glycine Pathway in Cupriavidus Necator. Metabolic Engineering 2020, 62, 30- 41. doi.org / 10.1016 / i.ymben.2020.08.004.Example 5: Growth-coupled microbial production of an animal pigment

[0207] Modern automated bioengineering platforms have been successfully employed to achieve the heterologous production of natural and engineered products, such as biofuels, drugs, and food ingredients, yet current research and developmental approaches are labor, resource, and time intensive. Herein we introduce a flexible, growth-coupled bioengineering method to efficiently overproduce molecules in engineered cells by linking production to life. We designed a Pseudomonas putida 5,10-methylenetetrahydrofolate (MTHF) auxotroph that is relieved by endogenously produced formate as a biosynthetic byproduct. By installing the versatile tryptophan- to-kynurenine pathway that creates a molecule of formate per each kynurenine-based product, we demonstrate the potential of the growth-coupled biosynthetic approach with the overproduction of the kynurenine-based dimer xanthommatin, a broad-spectrum, color-changing pigment from the animal kingdom with material and cosmetic potential. We used adaptive laboratory evolution to streamline the P. putida expression host to produce xanthommatin solely from glucose at over lOOx the amount from conventional overexpression approaches, thereby establishing growth-coupling as apromising biotechnological approach for establishing and optimizing the microbial production of value-added molecules.

[0208] Heterologous pathways, however, are not essential for life and place a large metabolic burden on the host while conferring no survival advantage. We imagined that converting a heterologous pathway into an essential one by directly tying bioproduction of an intentional product to cell survival would force metabolic flux through the pathway and thereby readily expedite higher production levels (FIG. 17A). The premise of this growth-coupled biosynthesis approach takes advantage of the observation that most metabolic pathways are not atom efficient and often coproduce byproducts en route to the ultimate metabolic product. We reasoned that if we could rewire a cell’s metabolism to become reliant upon a heterologously produced byproduct, then we could fully connect bioproduction to growth. As a proof of principle experiment for the bioengineering potential of this generalizable approach to sustainable products, we report here the creation of a bacterial host dependent for growth upon endogenously produced formate formed during the biosynthesis of the color-changing animal pigment xanthommatin (FIGS. 17B-17C).

[0209] The most common carbon-based metabolic byproducts are one-carbon (Cl) molecules, such as CO2, formate, and formaldehyde, produced during wide-ranging enzymatic reactions common in cellular metabolism. These Cl molecules are generally secreted or poorly assimilated back into central metabolism. In recent years, seminal progress has been made towards engineering model, industrially relevant microorganisms for growth on these very same Cl compounds as feedstocks. This has been accomplished by rewiring metabolism to introduce synthetic auxotrophies that are relieved by assimilation of exogenous Cl compounds (e g., Cl metabolites) whereupon they enter central metabolism via a series of interconnecting metabolic pathways via methionine and folate cycles. These essential cycles support purine and thymidine synthesis and the remethylation of homocysteine. Herein, we designed a Pseudomonas putida formate auxotroph in which we added a tryptophan-to-kynurenine biosynthetic pathway that releases one equivalent of endogenously produced formate per kynurenine molecule formed. We demonstrate the plasticity and industrial potential of the growth-coupled biosynthetic approach with the overproduction of the kynurenine- based dimer xanthommatin (Xa), a broad- spectrum color-changing biopigment from the animal kingdom. This biopigment plays important biological roles in camouflage, color-patterning,compound eye vision, and nuptial coloration in cephalopods and arthropods (FIG. 17B), and due to its optoelectronic properties, Xa has strong potential to be leveraged as a biomaterial, with potential applications ranging from photoelectronic devices, thermal management coatings, dyes, and UV- protectants. While xanthommatin can be obtained via organic extraction from animal tissues or chemical synthesis, processes which are low yielding and suffer in purity and scalability, our growth-coupled strategy was paramount to overproduce Xa for the first time in a microbe.

[0210] Results

[0211] Computational design and construction of a MTHF auxotroph. To explore the bioengineering potential of a growth-coupled biosynthesis system linked to Cl metabolism, we first designed a platform strain that could be broadly employed irrespective of the product as long as an appropriate C 1 byproduct is released. We selected the industrial strain Pseudomonas putida as a bacterial chassis for its versatile metabolism and high tolerance to toxic substrates and products, paired with resistance to oxidative stress. In our approach, we engineered a synthetic formate auxotrophy in P. putida modeled after a recent design in Escherichia coh. and then second, we transplanted a kynurenine biosynthetic pathway into the bacterial chassis to restore growth and facilitate product synthesis (FIG. 18).

[0212] Using genome-reduced P. putida EM42 as the parent strain, a derivative of KT2440 optimized for heterologous expression, we targeted the biosynthetic reconfiguration of the essential cofactor 5,10-methylenetetrahydrofolate (MTHF). In P. putida, MTHF is formed by the transfer of Cl units to THF from serine and glycine via serine hydroxymethyltransferase (SHMT, encoded by gfyA-I and -II) and the glycine cleavage system (encoded by gcvTHP-I and -II), respectively (FIG. 19). Moreover, MTHF is also a precursor for 10-formyl-THF and purine biosynthesis in P. putida. To restore MTHF biosynthesis, we employed the formate assimilation pathway from Methylobacteriuni extorquens that transfers formate directly to THF before being ultimately converted to MTHF. We first interrogated the formate auxotrophy design in silico using the genome-scale metabolic model iJN1463 for P. putida and the growth-coupling program algorithm gcOpt that predicted the deletion of gcvTHP and glyA (FIGS. 22A-22C). The flux space spanned by the metabolic model of the double-deletion strain shows a strict formate auxotrophy, whichcorresponds to a positive lower formate uptake bound for any given growth state (FIGS. 22A-22C). The respective metabolic flux rearrangements caused by the design channel THF-activated formate towards purine biosynthesis via inosine 5-monophosphate (IMP, 87% of synthesized formate), as well as L-methionine (12%) and pyrimidine (1%) biosynthesis via MTHF (FIGS. 22A-22C). The model also identified non-validated genes that, in theory, could also function to synthesize formyl- THF from formate such as phosphoribosyl glycinamide formyltransferase (PurT) and THF- dependent phosphoribosylglycinamide formyltransferase (PurN), and as such we maintained purT / purN in our design.

[0213] We built the P. putida synthetic formate auxotroph strain LBB05 by deleting both copies of glyA and the two gcvTHP operons (FIG. 18A). We also inserted an optimized formate assimilation operon, consisting of formate-THF ligase (FtfL, UniProt: Q83WS0), methenyl-THF cyclohydrolase (Fch, UniProt: Q49135), and methylene-THF dehydrogenase (MtdA, UniProt: P55818), into the pha locus, thereby deleting the native genes involved in polyhydroxyalkanoate biosynthesis. All genome modifications were carried out using Scel-assisted homologous recombination and, when necessary, CRISPR-Cas9 countersei ection. Following construction, we tested growth of strain LBB05 in minimal media under selective conditions. When only glucose was included as a carbon source, no growth was observed. In the presence of glucose and exogenous formate, only slight growth was detected. We suspected that endogenous levels of glycine due to the glyA deletion could be limiting due to the deletion of SHMT. However, metabolic models of the wildtype and LBB05 both reach the same maximum growth rates without cofeeding glycine, which suggests that the metabolic capacity to synthesize glycine is not impaired in LBB05, and points to an unfavorable regulatory regime instead (FIGS. 22A-22C). When exogenous glycine was further included in the media, robust growth was restored (FIG. 18B). We monitored the concentration of formate during growth and observed that formate consumption correlated with growth (FIG. 18C) and, moreover, that growth rate and maximum cell density achieved were dependent upon formate concentration (FIG. 18D).

[0214] Growth-coupled biosynthesis with endogenously produced formate from the kynurenine pathway. To test whether endogenously produced formate could also rescue growth and at the same time empower heterologous biosynthesis, we selected the kynurenine pathway inwhich C-2 of tryptophan is oxidatively removed as formate (FIG. 19A). The kynurenine pathway leads to diverse metabolic products with important roles in animals and is also active in pseudomonads such as P. aeruginosa and P. fluorescens that produce anthranilate and the siderophore thioquinolobactin, respectively. We sourced genes from the fluorescent pseudomonad Pseudomonas sp. DTU12.1, which harbors an active thioquinolobactin biosynthetic gene cluster (fibs)' , to construct two plasmid-based pathways for the synthesis of kynurenine and 3- hy droxy kynurenine .

[0215] The simplest construct consisted of two genes, namely qbsF and qbsH, that encode tryptophan 2,3 -dioxygenase (TDO) and kynurenine formamidase (KF A), respectively, to convert tryptophan to N-formylkynurenine and ultimately kynurenine plus formate (FIG. 19B). Upon introduction of qbsFHivAo the formate auxotroph to create strain LBB06, we evaluated its growth under selective conditions. We observed that tryptophan could replace formate to sustain growth only in LBB06, but not LBB05 (FIG. 19C), suggesting that MTHF biosynthesis was supported exclusively by the kynurenine pathway and not by native host pathways functioning as escape routes. Significantly, higher concentrations of tryptophan resulted in faster growth rate, higher maximum cell density, and in the overproduction of kynurenine and kynurenic acid, the latter of which is a likely product of an endogenously produced transaminase (FIGS. 19C-19D). We measured forty-five-fold and forty-four-fold increases in production at 72 h of growth of kynurenine and kynurenic acid, respectively, in LBB06 in comparison to the parent strain EM42 in which we similarly introduced the qbsFH gene cassette (LBB11) (FIG. 23), supporting the metabolic boost of the growth-coupled approach.

[0216] We similarly constructed a three-gene pathway to 3-hydroxykynurenine that further included qbsG encoding kynurenine monooxygenase (KMO). The resulting strain LBB07 was also dependent upon tryptophan and glycine for growth (FIG. 19C). In this case, we observed the overproduction of 3 -hydroxykynurenine (43.1 mg / L) and its transaminated product xanthurenic acid (10.1 mg / L), in addition to kynurenine (29.5 mg / L) and kynurenic acid (58.1 mg / L) after 3 days of cultivation, each of which were produced in significantly greater amounts than in EM42 expressing qbsFGH (LBB12) (FIG. 19D). Significantly, we observed profound color changes from yellow to orange during the growth of LBB07, but not in LBB12 (FIG. 19E), that were not due to thekynurenine-based monomers. HPLC-MS analysis revealed the formation of two chromophores with absorption at 440 nm (FIG. 19D) that we isolated and characterized by NMR as the 3- hydroxykynurenine-based dimers xanthommatin and decarboxylated xanthommatin at approximately 154.3 mg / L (FIG. 23). We treated the isolated xanthommatin compounds with the reductant ascorbic acid and the oxidant sodium nitrite and observed the anticipated redox-dependent color-changes (FIGS. 24A-24B) as previously described. Thus, despite not including an exogenous dimerase gene, which has yet to be experimentally established in animals, the overexpression of the qbsFGH genes resulted in the unexpected synthesis of the animal pigment xanthommatin in LBB07.

[0217] Adaptive laboratory evolution (ALE) to streamline growth-coupled biosynthesis.Despite the metabolic advantage of strains LBB06 and LBB07 in producing kynurenine molecules, those strains require supplemental glycine and tryptophan for growth as confirmed by the metabolic model. Yet the cells have endogenous routes to synthesize these amino acids from glucose. We thus applied adaptive laboratory evolution (ALE) to successfully wean the system off these supplemental amino acids. We first subjected the formate auxotroph LBB05 to ALE to support growth in the absence of exogenously supplied glycine (FIGS. 26A-26H). We planned to wean the strain off glycine by decreasing the concentration of glycine stepwise with each passage. At each passage point, cells are also passaged into a corresponding ‘test’ flask containing no glycine to test whether the currently growing population has been weaned off Gly. Once growth is observed in the ‘test’ flask, a minimum of 2 more passages in glycine-free medium will be carried out. To our surprise, all eight replicate experiments showed growth in the very first test flask, making the stepwise weaning protocol unnecessary. A single isolate was obtained from five of the eight endpoint populations, its growth profiled and then subjected to whole genome sequencing. All five isolates contained missense mutations in the gene metK, which codes for S-adenosylmethionine synthase, yet at different sequence locations. No other genomic mutations were found in all strains, implicating the unexpected role of metK activity in glycine metabolism and revealing the power of growth-coupled ALE to enhance strain platforms not predicted by rational-engineering based approaches. We next tested the prevalence and diversity of MetK mutants within a single population (A8-F3-I0), selected at random. A library of metK amplicons was generated by PCR and subjected to ONT sequencing. Each amino acid position was then examined for the percentage of amplicons containing a certainmutation at that position. 17 mutations were found at a frequency of greater than 1%. The top three most frequent were R281, P237T, and H35OP (FIG. 20G).

[0218] Next, we transferred the kynurenine (qbsFH) and 3-hydroxykynurenine (qbsFGH) gene cassettes into evolved population A8-F3-I0. We opted to go ahead with a population rather than a clonal isolate to fully sample the metK sequence space and avoid bias towards a single metK mutant. As expected, transformants produced their respective kynurenine pathway products without supplemental glycine, yet still required tryptophan-feeding for growth in minimal medium, indicating that the metK mutations were maintained. We then carried out another round of ALE to remove the tryptophan dependency following the same weaning protocol as with the glycine requirement (FIGS. 27A-27J). Five distinct qbsFH and five distinct qbsFGH transformants were selected to initiate parallel evolution experiments. The qbsFH experiments, similar to glycine, exhibited growth in the first test flask as did two of the qbsFGH experiments. For the other three qbsFGH experiments, the concentration of L-Trp was decreased four-fold before growth was observed in the test flask. Five isolated clones from each of the endpoint population were characterized. All grew in Trp-free minimal minimum and showed similar production profiles.

[0219] To evaluate the genomic changes in the evolved glycine / tiyptophan-independent strains, we selected one clone per population for whole-genome sequencing. Strikingly, none of the apparent genomic mutations were common to all clones. The pathway plasmids, however, all contained mutations in the PEM? promoter region, ranging from deletions to insertional repeats. To test the consequence of modified P EM7, we isolated plasmids from some of the evolved clones, and introduced them directly into hosts LBB05 and LBB08. The resulting strains were indeed able to grow in Trp-free minimal media, indicating that the modified promoters are sufficient to impart Trp- independent growth.

[0220] Exemplary Bioproduction Strain

[0221] We conducted a time course study in shake flasks with two of the evolved clones A2-F3-I3 (LBB \ 3-qbsFH) and A18-F4-I3 (LBB14-qbsFGH), comparing production levels to EM42 strains LBB11 and LBB12, respectively, with glucose as the sole carbon source. We again measured enhanced metabolic production in the kynurenine / kynurenic acid-producing strain LBB13 at levels(59.8 mg / L kynurenine and 181.2 mg / L kynurenic acid) on par with unevolved LBB06 that required supplemental glycine and tryptophan (FIGS. 29A-29D). The 3-hydroxykynurenine producing strain LBB14, on the other hand, exhibited a beneficial shift in its metabolite profile with a decrease in the amount of the shunt product kynurenic acid relative to the amount of L-3 -hydroxykynurenine. Xa was produced by LBB14 when grown with glucose as the sole carbon source, but not by LBB12, as was the case even with Trp feeding. The quantities of the other metabolites were also greater in LBB14, with twenty-eight, eleven, thirty-one, and twelve-fold increases in kynurenine, kynurenic acid, hydroxykynurenine, and xanthurenic acid, respectively.

[0222] Discussion

[0223] Xanthommatin is a redox-active color-changing biopigment from the animal kingdom. Its special properties make it an attractive sustainable biomaterial but obtaining it from natural animal sources or by chemical synthesis presents challenges. In lieu of following an iterative stepwise traditional process of engineering a microbial production strain, we re-programmed host metabolism making biosynthesis of the kynurenine monomer mandatory for growth. Our growth-coupling approach activated the heterologous pathway in minimal medium, and unexpectedly, drove dimerization of the L-3 -hydroxykynurenine monomer, even though no dedicated dimerase was installed in the pathway. The growth-coupling scheme provided a tight selection that facilitated rapid, reproducible adaptive laboratory evolution, yielding an engineered strain capable of gramscale production in a minimal medium under fed-batch conditions. Here, we created the first microbial bioproduction platform to xanthommatin, a scalable, sustainable alternative to chemical synthesis and extraction from animal tissues. Further, we devised a facile purification route, in which xanthommatin is precipitated directly from aqueous medium, by addition of a biological reductant. This opens doors towards developing it for applications in materials and skincare and as heterologous platform for production of designer xanthommatin analogs with novel properties.

[0224] While we have focused here on xanthommatin, the auxotrophic host strain we engineered has the potential to function with any pathway that releases a one-carbon byproduct - i.e. formate, formaldehyde, methanol, cyanide, even CO2. One-carbon byproducts are ubiquitous in metabolicpathways, across a wide range of enzymatic classes, making the platform strain suitable for high yield production of many chemical products.

[0225] Growth -coupling has mainly been explored in the context of endogenously produced central metabolites (I.e. ethanol..) We found that using an auxotrophic background resulted in higher production levels under selective conditions relative to wildtype at a fold-improvement in a timeframe inaccessible to traditional bioengineering platforms. This study showcases the power of auxotrophic backgrounds hosts to improve production at a fold-improvement in a timeframe inaccessible to traditional bioengineering platforms.Example 6: Growth rescue in vanAB-expressing bacterial cells

[0226] Electrocompetent P. putida LBB08 was transformed with plasmids pS24Pem7(BCD2)- vanAB and pS24Pem7(BCD2)-gfp and plated out on to LB agar plates containing 50 pg ml'1kanamycin and incubated at 30 °C. Single colonies were cultured in 3 mL of LB media containing 50 pg ml’1kanamycin at 30 °C for 16 h with shaking at 250 rpm. 90 pL of the overnight culture was used to inoculate a fresh culture tube containing 3 mL de Bont minimal media supplemented with 20 mM glucose, 10 mM sodium formate and 50 pg ml’1kanamycin. The culture was grown at 30 °C for 16 h with shaking at 250 rpm. Cells were washed 3x ImL with de Bont minimal media supplemented with 20 mM glucose and normalized to OD600 = 0.2. 50 pL of cells were transferred to 96-well microtiter plates containing 2 mM vanillic acid in 50 pL de Bont minimal media supplemented with 20 mM glucose, 100 pg ml’1kanamycin and 2% DMSO cosolvent. Cell growth was monitored at 600 nm absorbance over 30 hours using a TECAN spark plate reader (FIG. 30).P EMBODIMENTS

[0227] P Embodiment 1. A cell, wherein the cell comprises an auxotroph.

[0228] P Embodiment 2. The cell of P Embodiment 1, wherein the auxotroph comprises a 1- carbon (Cl) metabolite auxotroph.

[0229] P Embodiment 3. The cell of P Embodiment 1 or 2, wherein the cell is unable to synthesize a cofactor in the absence of the Cl metabolite.

[0230] P Embodiment 4. The cell of any one of P Embodiments 1-3, wherein the cell is capable of synthesizing a biomolecule.

[0231] P Embodiment 5. The cell of any one of P Embodiments 1-4, wherein the cell does not comprise a glyA gene or a gcvTHP gene.

[0232] P Embodiment 6. The cell of P Embodiment 5, wherein the glyA gene encodes a serine hydroxymethyltransferase protein.

[0233] P Embodiment 7. The cell of P Embodiment 5, wherein the gcvTHP gene encodes enzymes of the glycine cleavage system.

[0234] P Embodiment 8. The cell of any one of P Embodiments 1-7, wherein the cell comprises a formate-THF ligase protein (FtfL), a methenyl-THF cyclohydrolase protein (Fch), and / or a methylene-THF dehydrogenase protein (MtdA).

[0235] P Embodiment 9. The cell of any one of P Embodiments 3-8, wherein the cofactor comprises 5,10-methylene-THF (MTHF).

[0236] P Embodiment 10. The cell of any one of P Embodiments 1-9, wherein the Cl metabolite comprises a formate, a formaldehyde, a methanol, a methylamine, a methane, a carbon monoxide, and / or a carbon dioxide.

[0237] P Embodiment 11. The cell of any one of P Embodiments 2-10, wherein the auxotroph further comprises a glycine auxotroph.

[0238] P Embodiment 12. The cell of P Embodiment 11, wherein in the cell does not comprise aItaE gene.

[0239] P Embodiment 13. The cell of P Embodiment 12, wherein the ItaE gene encodes L- threonine aldolase protein.

[0240] P Embodiment 14. The cell of any one of P Embodiments 1-13, further comprising a tryptophan dioxygenase protein (TDO), a kynurenine formamidase protein (KF A), and / or a kynurenine monooxygenase protein (KMO).

[0241] P Embodiment 15. The cell of P Embodiment 14, wherein the cell synthesizes a 3- hydroxykynurenine (3-HK).

[0242] P Embodiment 16. The cell of P Embodiment 15, wherein the 3-HK is dimerized by oxidative cyclization.

[0243] P Embodiment 17. ’ The cell of P Embodiment 16, wherein the oxidative cyclization comprises a heme-peroxidase.

[0244] P Embodiment 18. The cell of any one of P Embodiments 14-17, wherein the biomolecule comprises a xanthommatin (Xa).

[0245] P Embodiment 19. The cell of any one of P Embodiments 1-18, wherein the cell comprises a bacterial cell.

[0246] P Embodiment 20. The cell of P Embodiment 19, wherein the bacterial cell comprises aPseudomonas putida cell.

[0247] P Embodiment 21. The cell of P Embodiment 20, wherein the Pseudomonas putida cell comprises a Pseudomonas putida KT2440 cell

[0248] P Embodiment 22. A method for synthesizing a biomolecule, the method comprising: (a) culturing the cell of any one of P Embodiments 1-21; (b) allowing the cell to synthesize the biomolecule.

[0249] P Embodiment 23. The method of P Embodiment 22, wherein the synthesis of the biomolecule is coupled to cell growth and / or survival.

[0250] P Embodiment 24. The method of P Embodiment 22 or 23, wherein the biomolecule comprises 5,10-methylene-THF (MTHF), 3 -hydroxy kynurenine (3-HK), xanthommatin (Xa), and / or methyl anthranilate.EMBODIMENTS

[0251] Embodiment 1. A bacterial cell comprising a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase(MtdA) protein, wherein the bacterial cell does not express aglyA gene product and agcvTHP gene product, and wherein the bacterial cell endogenously produces a formate.

[0252] Embodiment 2. The bacterial cell of Embodiment 1, wherein all glyA genes and all gcvTHP genes are knocked out.

[0253] Embodiment 3. The bacterial cell of Embodiment 1 or 2, further comprising a formamidase protein, cyanide dihydratase protein, a formate dehydrogenase protein, or a formaldehyde dehydrogenase protein.

[0254] Embodiment 4. The bacterial cell of any one of Embodiments 1-3, further comprising a cyanide hydratase protein, a carbon monoxide dehydrogenase protein, a carbonic anhydrase protein, a dichloromethane dehalogenase protein, a methanethiol oxidase protein, a methanesulfonate monooxygenase protein, a methanol dehydrogenase protein, or a methylamine dehydrogenase protein.

[0255] Embodiment 5. The bacterial cell of Embodiment 4, further comprising a methane monooxygenase protein.

[0256] Embodiment 6. The bacterial cell of any one of Embodiments 1-5, further comprising a tryptophan dioxygenase (TDO) protein and a kynurenine formamidase (KF A) protein.

[0257] Embodiment 7. The bacterial cell of Embodiment 6, wherein the bacterial cell produces an L-3 -hydroxy kynurenine.

[0258] Embodiment 8. The bacterial cell of Embodiment 6, further comprising a kynurenine monooxygenase (KMO) protein.

[0259] Embodiment 9. The bacterial cell of Embodiment 8, wherein the cell produces a xanthommatin (Xa).

[0260] Embodiment 10. The bacterial cell of any one of Embodiments 1-9, further comprising a kynurenine aminotransferase protein, a kynureninase protein, or an aromatic L-amino acid decarboxylase protein.

[0261] Embodiment 11. The bacterial cell of Embodiment 10, wherein the cell produces a kynurenic acid, an anthranilic acid, a kynuramine, a xanthurenic acid, a 3-hydroxyanthranilic acid, a cinnabarinic acid, or a 3-hydroxykynuramine.

[0262] Embodiment 12. The bacterial cell of Embodiment 10 or 11, further comprising a 3- hydroxyanthranilic acid oxidase protein or a 3-hydroxyanthranilic acid oxygenase protein.

[0263] Embodiment 13. The bacterial cell of Embodiment 12, wherein the cell produces a quinolinic acid or a picolinic acid.

[0264] Embodiment 14. The bacterial cell of any one of Embodiments 1-9, wherein the bacterial cell is a Pseudomonadota bacterial cell.

[0265] Embodiment 15. The bacterial cell of any one of Embodiments 1-14, wherein the bacterial cell is an Acidithiobacillia bacterial cell, an Alphaproteobacteria bacterial cell, a Betaproteobacteria bacterial cell, a Gammaproteobacteria bacterial cell, a Hydrogenophilia bacterial cell, or a Zetaproteobacteria bacterial cell.

[0266] Embodiment 16. The bacterial cell of any one of Embodiments 1-15, wherein the bacterial cell is a Pseudomonas bacterial cell.

[0267] Embodiment 17. The bacterial cell of Embodiment 16, wherein the bacterial cell is a Pseudomonas putida cell.

[0268] Embodiment 18. The bacterial cell of Embodiment 17, wherein the Pseudomonas putida cell is a Pseudomonas putida KT2440 cell.

[0269] Embodiment 19. The bacterial cell of any one of Embodiments 1-13, wherein the bacterial cell is a Bacillus bacterial cell.

[0270] Embodiment 20. The bacterial cell of Embodiment 19, wherein the Bacillus bacterial cell is a Bacillus subtilis bacterial cell.

[0271] Embodiment 21. The bacterial cell of any one of Embodiments 1-13, wherein the bacterial cell is a Actinomycetota bacterial cell.

[0272] Embodiment 22. The bacterial cell of Embodiment 21, wherein the Actinomycetota bacterial cell is a Streptomyces bacterial cell.

[0273] Embodiment 23. The bacterial cell of Embodiment 22, wherein the Streptomyces bacterial cell is a Streptomyces coelicolor bacterial cell.

[0274] Embodiment 24. A bacterial cell comprising a xanthommatin (Xa).

[0275] Embodiment 25. The bacterial cell of Embodiment 24, comprising a formate- tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein.

[0276] Embodiment 26. The bacterial cell of Embodiment 24 or 25, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product.

[0277] Embodiment 27. The bacterial cell of any one of Embodiments 24-26, further comprising a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and / or a kynurenine monooxygenase (KMO) protein.

[0278] Embodiment 28. The bacterial cell of any one of Embodiments 24-27, wherein the bacterial cell is applied to a surface or a hydrogel.

Claims

WHAT IS CLAIMED IS:

1. A bacterial cell comprising a formate-tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product, and wherein the bacterial cell endogenously produces a formate.

2. The bacterial cell of claim 1, wherein all glyA genes and all gcvTHP genes are knocked out.

3. The bacterial cell of claim 1, further comprising a formamidase protein, cyanide dihydratase protein, a formate dehydrogenase protein, or a formaldehyde dehydrogenase protein.

4. The bacterial cell of claim 3, further comprising a cyanide hydratase protein, a carbon monoxide dehydrogenase protein, a carbonic anhydrase protein, a dichloromethane dehalogenase protein, a methanethiol oxidase protein, a methanesulfonate monooxygenase protein, a methanol dehydrogenase protein, or a methylamine dehydrogenase protein.

5. The bacterial cell of claim 4, further comprising a methane monooxygenase protein.

6. The bacterial cell of claim 1, further comprising a tryptophan dioxygenase (TDO) protein and a kynurenine formamidase (KF A) protein.

7. The bacterial cell of claim 6, wherein the bacterial cell produces an L-3- hydroxy kynurenine.

8. The bacterial cell of claim 6, further comprising a kynurenine monooxygenase (KMO) protein.

9. The bacterial cell of claim 8, wherein the cell produces a xanthommatin(Xa).

10. The bacterial cell of claim 1, further comprising a kynurenine aminotransferase protein, a kynureninase protein, or an aromatic L-amino acid decarboxylase protein.

11. The bacterial cell of claim 10, wherein the cell produces a kynurenic acid, an anthranilic acid, a kynuramine, a xanthurenic acid, a 3-hydroxyanthranilic acid, a cinnabarinic acid, or a 3-hydroxykynuramine.

12. The bacterial cell of claim 10, further comprising a 3-hydroxyanthranilic acid oxidase protein or a 3-hydroxyanthranilic acid oxygenase protein.

13. The bacterial cell of claim 12, wherein the cell produces a quinolinic acid or a picolinic acid.

14. The bacterial cell of any one of claims 1, wherein the bacterial cell is a Pseudomonadota bacterial cell.

15. The bacterial cell of claim 1, wherein the bacterial cell is an Acidithiobacillia bacterial cell, an Alphaproteobacteria bacterial cell, a Betaproteobacteria bacterial cell, a Gammaproteobacteria bacterial cell, a Hydrogenophilia bacterial cell, or a Zetaproteobacteria bacterial cell.

16. The bacterial cell of claim 1, wherein the bacterial cell is a Pseudomonas bacterial cell.

17. The bacterial cell of claim 16, wherein the Pseudomonas bacterial cell is a Pseudomonas putida cell.

18. The bacterial cell of claim 17, wherein the Pseudomonas putida cell is a Pseudomonas putida KT2440 cell.

19. The bacterial cell of claim 1, wherein the bacterial cell is a Bacillus bacterial cell.

20. The bacterial cell of claim 19, wherein the Bacillus bacterial cell is a Bacillus subtilis bacterial cell.

21. The bacterial cell of claim 1, wherein the bacterial cell is a Actinomycetota bacterial cell.

22. The bacterial cell of claim 21, wherein the Actinomycetota bacterial cell is a Streptomyces bacterial cell.

23. The bacterial cell of claim 22, wherein the Streptomyces bacterial cell is a Streptomyces coelicolor bacterial cell.

24. A bacterial cell comprising a xanthommatin (Xa).

25. The bacterial cell of claim 24, comprising a formate- tetrahydrofolate(THF) ligase (FtfL) protein, a methenyl-THF cyclohydrolase (Fch) protein, and a methylene-THF dehydrogenase (MtdA) protein.

26. The bacterial cell of claim 24, wherein the bacterial cell does not express a glyA gene product and a gcvTHP gene product.

27. The bacterial cell of claim 24, further comprising a tryptophan dioxygenase (TDO) protein, a kynurenine formamidase (KF A) protein, and / or a kynurenine monooxygenase (KMO) protein.

28. The bacterial cell of claim 24, wherein the bacterial cell is applied to a surface or a hydrogel.

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