WO2017136795A1 - Bacteria engineered to treat diseases associated with tryptophan metabolism - Google Patents

Abstract

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with dysregulated tryptophan metabolism are disclosed.

Description

Bacteria Engineered to Treat Diseases Associated with Tryptophan

Metabolism

Related Applications

[01] The instant application claims priority to United States Provisional Patent Application No. 62/291,461, filed February 4, 2016; United States Provisional Patent Application No. 62/291,468, filed February 4, 2016; United States Provisional Patent Application No. 62/291,470, filed February 4, 2016; United States Provisional Patent Application No. 62/297,778, filed February 19, 2016; International Application No. PCT/US2016/020530, filed March 2, 2016; United States Provisional Patent Application No. 62/305,462, filed March 8, 2016; United States Provisional Patent Application No. 62/313,691, filed March 25, 2016; United States Provisional Patent Application No. 62/314,322, filed March 28, 2016; United States Provisional Patent Application No. 62/335,940, filed May 13, 2016; International Application No.

PCT/US2016/032565, filed May 13, 2016; United States Provisional Patent Application No. 62/347,508, filed June 8, 2016; United States Provisional Patent Application No. 62/347,576, filed June 8, 2016; United States Provisional Patent Application No.

62/348,620, filed June 10, 2016; United States Provisional Patent Application No.

62/348,360, filed June 10, 2016; United States Provisional Patent Application No.

62/354,682, filed June 24, 2016; International Application No. PCT/US2016/039444, filed June 24, 2016; United States Provisional Patent Application No. 62/362,954, filed July 15, 2016; United States Provisional Patent Application No. 62/385,235, filed September 8, 2016; International Application No. PCT/US2016/050836, filed

September 8, 2016; United States Patent Application No. 15/260,319, filed September 8, 2016; United States Provisional Patent Application No. 62/423,170, filed November 16, 2016; United States Provisional Patent Application No. 62/439,871, filed December 28, 2016; International Application No. PCT/US2016/069052, filed December 28, 2016; and United States Provisional Patent Application No. 62/443,639, filed January 6, 2017, the entire contents of each of which are expressly incorporated herein by reference in their entireties.

Background

[02] Tryptophan (TRP) is an essential amino acid that, after consumption, is either incorporated into proteins via new protein synthesis, or converted a number of biologically active metabolites with a number of differing roles in health and disease (Perez-De La Cruz et ah, 2007 Kynurenine Pathway and Disease: An Overview;

CNS&Neurological Disorders -Drug Targets 2007, 6,398-410). These metabolites, along with the enzymes responsible for their production, have implications in a broad range of diseases, including, but not limited to, infectious disease, cancer and the immune escape, various neurological conditions, autoimmune disease, such as IBD, and metabolic diseases, including but not limited to, NASH, diabetes, and obesity.

[03] Along one arm of tryptophan catabolism, trytophan is converted to the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) in select populations of neurons by tryptophan hydroxylase. Serotonin can further be converted into the hormone melatonin. The majority of tryptophan, approximately 95%, however, is metabolized to a number of bioactive metabolites, collectively called kynurenines, along a second arm called the kynurenine pathway (KP). In the first step of catabolism, TRP is converted to Kynurenine, (KYN), which has well-documented immune suppressive functions in several types of immune cells, and has recently been shown to be an activating ligand for the arylcarbon receptor (AhR; also known as dioxin receptor). KYN may be further a number of downstream bioactive metabolites, which inter alia have immunosuppressive, neuroprotective and neurotoxic roles.

[04] More recently, a number of tryptophan metabolites, which are produced by the by bacteria in the gut or taken up by the diet, such as indole-3 aldehyde, indole-3 acetate, or indole-3 propionic and many others, collectively termed "indoles", herein, also have been shown to be ant i- inflammatory and protective of gut-barrier function, mediated through AhR agonism.

[05] Therefore, finding a means to upregulate and/or downregulate the levels of tryptophan and its metabolites and to modify relative amounts and/or ratios of tryptophan and its various bioactive metabolites is useful in the prevention, treatment and/or management of a number of disease as described herein. The present disclosure describes compositions for regulating and fine tuning levels and/or ratios of tryptophan and its metabolites and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.

[06] This disclosure relates to compositions and therapeutic methods for treating diseases or disorders with immunosuppressive and/or inflammatory

components, by altering levels tryptophan and/or its metabolites and/or by alterning relative amounts and/or ratios of tryptophan and its various bioactive metabolites. In certain aspects, the disclosure relates to compositions, methods, and uses of engineered bacteria that are capable producing and/or consuming one or more tryptophan metabolites. In some embodiments, the engineered bacteria are capable of reducing inflammation in the gut and/or enhancing gut barrier function, and thereby ameliorating or preventing an autoimmune, metabolic, and/or a neurological disorder, and/or viral infection. In other aspects, the present disclosure provides compositions, methods, and uses of genetically engineered bacteria that produce and/or consume one or more tryptophan metabolites that selectively target tumors and tumor cells, for the treatment and/or prevention of cancer.

Summary

[07] The disclosure provides genetically engineered bacteria that are capable of modulating levels of tryptophan and its metabolites. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to alter tryptophan to tryptophan metabolite ratios or to modulate systemic tryptophan and/or tryptophan metabolite availability.

[08] In certain embodiments, the genetically engineered bacteria are nonpathogenic and may be introduced into the gut in order to increase kynurenine.

[09] In certain embodiments, the genetically engineered bacteria are nonpathogenic and may be introduced into the gut in order to decrease tryptophan.

[010] In some embodiments the disclosure provides a genetically engineered bacterium comprising one or more gene sequences for the modulation of tryptophan and/or tryptophan metabolites in the blood or gut of a mammal. In some embodiments, the tryptophan levels are increased in the blood or gut of a mammal. In some embodiments, the tryptophan levels are decreased in the blood or gut of a mammal. In some embodiments, the kynurenine levels are decreased in the blood or gut of a mammal. In some embodiments, the kynurenine levels are increased in the blood or gut of a mammal. In some embodiments, tryptamine levels are decreased in the blood or gut of a mammal.In some embodiemnts the tryptamine levels are increased in the blood or gut of a mammal. In some embodiments, indole-3-acetic acid levels are decreased in the blood or gut of a mammal. [Oi l] In some embodiments the disclosure provides a genetically engineered bacterium comprising at least one gene or gene cassette encoding one or more enzymes for the production of tryptophan. In some embodiments, the bacterium comprises gene sequence encoding TrpE. In some embodiments, the bacterium comprises gene sequence encoding feedback resistant TrpE. In some embodiments, the bacterium comprises gene sequence encoding trpDCBA. In some embodiments, the bacterium comprises gene sequence encoding aroG. In some embodiments, the bacterium comprises gene sequence encoding feedback resistant aroG (aroGfbr). In some embodiments, the bacterium comprises gene sequence encoding SerA. In some embodiments, the bacterium comprises gene sequence encoding feedback resistant SerA (SerAfbr). In some embodiments, the bacterium comprises an endogenous TnaA gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises an endogenous trpR gene which is knocked down via mutation or deletion.

[012] In some embodiments, the disclosure provides a genetically engineered bacterium comprising gene sequence for the degradation of kynurenine. In some embodiments, the bacterium comprises gene sequence encoding one or more

kynureninase polypeptide(s). In some embodiments, the bacterium comprises an endogenous trypE gene which is knocked down via mutation or deletion. In some embodiments, the bacterium an endogenous tyrB gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises gene sequence encoding one or more enzymes for the production of tryptophan.

[013] In some embodiments, the disclosure provides a bacterium comprising at least one gene or gene cassette encoding one or more enzymes for the production of tryptamine. In some embodiments, the bacterium comprises gene sequence encoding tryptophan decarboxylase (Tdc). In some embodiments, the bacterium comprises one or more gene sequence(s) encoding enzymes for the production of tryptophan.

[014] In some embodiments, the disclosure provides a genetically engineered bacterium comprising at least one gene or gene cassette encoding one or more enzymes for the production of indole- 3 -acetic acid. In some embodiments, the bacterium comprises gene sequence encoding tryptophan dehydrogenase (trpDH). In some embodiments, the bacterium comprises gene sequence encoding Indole-3-pyruvate decarboxylase (ipdC). In some embodiments the bacterium comprises gene sequence encoding Indole- 3 -acetaldehyde dehydrogenase (iadl). In some embodiments, the bacterium comprises gene sequence encoding enzymes for the production of tryptophan. In some embodiments, the bacterium is a thyA auxotroph. IN some embodiments, the gene or gene cassette encoding one or more enzymes for the production of tryptophan is operably linked to a directly or indirectly inducible promoter. In some embodiments, the promoter is induced by exogenous environmental conditions found in a mammalian gut. In some embodiments, the promoter is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is an FNR promoter selected from nirB L nirB2, nirB3, ydfZ, fnrS L and fnrS2. In some embodiments, the gene or gene cassette encoding one or more enzymes for the production of tryptophan is operably linked to a constitutive promoter. In some embodiments, the bacterium comprise one or more gene sequences encoding a gut barrier enhancer molecule and/or an antiinflammatory effector, e.g., selected from a short chain fatty acid, an ant i- inflammatory cytokine, Glp-2, IL-10 and IL-22. In some embodiments, the bacterium further comprises gene sequences encoding a checkpoint inhibitor and/or a pro-inflammatory cytokine, e.g., selected from anti-PD-1, anti-PD-Ll, anti-LAG3, anti-TIMl, anti- CTLA4 antibodies, and IL-15.

[015] In some embodiments, the disclosure provides a pharmaceutically acceptable composition comprising the genetically engineered bacterium of any one of claims 1-85 and a pharmaceutically acceptable carrier.

[016] In certain embodiments, the genetically engineered bacteria are nonpathogenic and may be introduced into the tumor microenvironment in order to reduced local kynurenine levels and/or increase tryptophan levels.

[017] Another aspect of the invention provides methods for selecting or targeting genetically engineered bacteria based on increased levels of kynureinin consumption. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with immune suppression and/or inflammation.

[018] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) or circuit(s), containing one or more native or non- native component(s), which mediate one or more mechanisms of action. Additionally, one or more endogenous genes or regulatory regions within the bacterial chromosome may be mutated or deleted. The genetically engineered bacteria harbor these genes or gene cassettes or circuits on a plasmid or, alternatively, the genes/gene cassettes have been inserted into the chromosome at certain regions, where they do not interfere with essential gene expression.

[019] These gene(s)/gene cassette(s) may be under the control of constitutive or inducible promoters. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by molecules or metabolites indicative of the gut or the tumor micorenvironment, promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and/or tetracycline. In some embodiments, the one or more gene sequences(s) are under the control of a constitutive promoter.

[020] In addition, the engineered bacteria may further comprise one or more of more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill- switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Brief Description of the Figures

[021] FIG. 1A, FIG. IB, FIG. 1C, and FIG. ID depicts schematics of exemplary embodiments, of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level- dependent promoters (e.g., FNR- inducible promoter), promoters induced by

inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g. , deletion of thyA (Δ thyA; thymidine dependence). FIG. 1A shows a schematic depicting an exemplary Tryptophan circuit. Tryptophan is produced from its precursor, chorismate, through expression of the trpE, trpG-D (also referred to as trpD), trpC-F (also referred to as trpC), trpB and trpA genes. Optional knockout of the tryptophan repressor trpR is also depicted. Optional production of chorismate through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. IB, and/or FIG. 1C, and/or FIG. ID. FIG. IB depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 1A and/or described in the description of FIG. 1A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 1C, and/or FIG. ID.

Optionally, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. FIG. 1C depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further comprises either a wild type or a feedback resistant SerA gene. Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD 1 to NADH. E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 1A and/or described in the description of FIG. 1A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. IB, and/or FIG. ID. Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. FIG. ID depicts a non-limiting example of a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further optionally comprises either a wild type or a feedback resistant SerA gene. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 1A and/or described in the description of FIG. 1A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. IB, and/or FIG. 1C. Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. Optionally, the bacteria may also comprise a deletion in PheA, which prevents conversion of chorismate into phenylalanine and thereby promotes the production of anthranilate and tryptophan.

[022] FIG. 2 depicts a schematic of tryptophan metabolism along the kynurenine and the serotonin arms in humans. The abbreviations for the enzymes are as follows: 3-HAO: 3-hydroxyl-anthranilate 3,4-dioxidase; AAAD: aromatic -amino acid decarboxylase; ACMSD, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase; HIOMT, hydroxyl-O-methyltransferase; IDO, indoleamine 2,3- dioxygenase; KAT, kynurenine amino transferases I-III; KMO: kynurenine 3- monooxygenase; KYNU, kynureninase; NAT, N-acetyltransferase; TDO, tryptophan 2,3-dioxygenase; TPH, tryptophan hydroxylase; QPRT, quinolinic acid phosphoribosyl transferase.

[023] FIG. 3 depicts a schematic of the E. coli tryptophan synthesis pathway. In Escherichia coli, tryptophan is biosynthesized from chorismate, the principal common precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine, as well as the essential compounds tetrahydrofolate, ubiquinone-8, menaquinone-8 and enterobactin (enterochelin), as shown in the superpathway of chorismate metabolism. Five genes encode five enzymes that catalyze tryptophan biosynthesis from chorismate. The five genes trpE trpD trpC trpB trpA form a single transcription unit, the trp operon. A weak internal promoter also exists within the trpD structural gene that provides low, constitutive levels of mRNA.

[024] FIG. 4 depicts a schematic of bacterial tryptophan catabolism

machinery, which is genetically and functionally homologous to IDOl enzymatic activity, as described in Vujkovic-Cvijin et al., Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism; Sci Transl Med. 2013 July 10; 5(193): 193ra91, the contents of which is herein incorporated by reference in its entirety. In certain embodiments, of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 4. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 4, including but not limited to, kynurenine, indole-3- aldehyde, indole- 3 -acetic acid, and/or indole-3 acetaldehyde.

[025] FIG. 5 depicts a schematic of the trypophan catabolic pathway/indole biosynthesis pathways. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al., CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016). In certain embodiments, of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 5 and FIG. 9. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 5 and FIG. 8 including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde.

[026] FIG. 6A and FIG. 6B depict diagrams of bacterial tryptophan metabolism pathways. FIG. 6A depicts a schematic of the bacterial tryptophan metabolism, as described, e.g., in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase (ECl.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2- monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0). The dotted lines ( ) indicate a spontaneous reaction. FIG. 6B Depicts a schematic of tryptophan derived pathways. Known AHR agonists are with asterisk. Abbreviations are as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole- 3 -acetaldehyde; IAA: Indole- 3 -acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA: Indole-3-pyruvic acid; IAM: Indole- 3 -acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3- acetonitrile; N-formyl Kyn: N-formylkynurenine;; Kyn:Kynurenine; KynA: Kynurenic acid; I3C: Indole-3-carbinol; IAld: Indole- 3 -aldehyde; DIM: 3,3'-Diindolylmethane; ICZ: Indolo(3,2-b)carbazole. Enzymes are numbered as follows: 1. EC 1.13.11.11 (Tdo2, Bna2), EC 1.13.11.11 (Idol); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4. EC 1.2.1.3 (ladl), EC 1.2.3.7 (Aaol); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclbl, Cclb2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125

(CYP79B2, CYP79B3); 9. EC 1.4.3.2 (StaO), EC 2.6.1.27 (Aro9, aspC), EC 2.6.1.99 (Taal), EC 1.4.1.19 (TrpDH); 10. EC 1.13.12.3 (laaM); 11. EC 4.1.1.74 (IpdC); 12. EC 1.14.13.168 (Yuc2); 13. EC 3.5.1.4 (IaaH); 14. EC 3.5.5.1. (Nitl); 15. EC 4.2.1.84 (Nitl); 16. EC 4.99.1.6 (CYP71A13); 17. EC 3.2.1.147 (Pen2). In certain embodiments, of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 6A and FIG. 6B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 6A and FIG. 6B. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments, the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[027] FIG. 7 depicts a schematic of disease states, in which correlative or causative roles for metabolites of the kynurenine pathway (KP) have been described.

[028] FIG. 8 depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease in the gut. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IP A) and Indole- 3 -aldehyde (I3A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function. I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL- 22 production. Activation of AhR plays a crucial role in gut immunity, such as in maintaining the epithelial barrier function and promoting immune tolerance to promote microbial commensalism while protecting against pathogenic infections. Indole has a number of roles, such as a signaling molecule to intestinal L cells to produce glucagon- like protein 1 (GLP-1) or as a ligand for AhR (Zhang et al. Genome Med. 2016; 8: 46).

[029] FIG. 9 depicts a schematic of one embodiment of the disclosure. In this embodiment, tryptophan is synthesized from kynurenine. Through this conversion, a immune- suppressive metabolite (kynurenine) can be removed from the external environment, e.g., a tumor environment, and a pro -inflammatory metabolite

(tryptophan) is generated. Kynureninase from Pseudomonas fluorescens converts KYN to AA (Anthranillic acid), which then can be converted to tryptophan through the enzymes of the E. coli trp operon. Optionally, the trpE gene may be deleted as it is not needed for the generation of tryptophan from kynurenine. In alternate embodiments, the trpE gene is not deleted, in order to maximize tryptophan production by using both kynurenine and chorismate as a substrate. In one embodiment of the invention, the genetically engineered bacteria comprising this circuit may be useful for reducing immune escape in cancer. In some embodiments, a new strain is generated through adaptive laboratory evolution. The ability of this strain to metabolize kynurenine is improved (through lowering of kynurenine substrate). Additionally, the ability or preference of the strain take up tryptophan is lowered (due to selection pressure imposed by toxic tryptophan analogs. As a result, this strain has improved therapeutic properties in a number of applications, including but not limited to immunoncology.

[030] FIG. 10 depicts a bar graph which shows the results of a checkerboard assay to establish the concentrations of kynurenine and 5-fluoro-L-tryptophan (ToxTrp) capable of sustaining growth of a trpE mutant of E. coli Nissle expressing

pseudoKYNase. Bacteria were grown in the presence of different concentrations of KYNU and ToxTrp, and in the absence of Anhydrous Tetracycline (aTc). Growth was assessed at OD600.

[031] FIG. 11 depicts a bar graph which shows the results of a checkerboard assay to establish the concentrations of kynurenine and 5-fluoro-L-tryptophan (ToxTrp) capable of sustaining growth of a trpE mutant of E. coli Nissle expressing

pseudoKYNase. Bacteria were grown in the presence of different concentrations of KYNU and ToxTrp, and in the presence of Anhydrous Tetracycline (aTc). Growth was assessed at OD600. [032] FIG. 12 depicts a bar graph which shows the growth of the wild-type E. coli Nissle (SYN094) and a control strain in which trpE is knocked out in M9+KYNU, without ToxTrp.

[033] FIG. 13 depicts a bar graph showing the kynurenine consumption rates of original and ALE evolved kynureninase expressing strains in M9 media

supplemented with 75 uM kynurenine. Strains are labeled as follows: SYN1404: E. coli Nissle comprising a deletion in Trp:E and a medium copy plasmid expressing kynureninase from Pseudomonas fluorescens under the control of a tetracycline inducible promoter (Nissle delta TrpE::CmR + Ptet-Pseudomonas KYNU pl5a KanR); SYN2027: E. coli Nissle comprising a deletion in Trp:E and expressing kynureninase from Pseudomonas fluorescens under the control of a constitutive promoter (the endogenous lpp promoter) integrated into the genome at the HA3/4 site (HA3/4::Plpp- pKYNase KanR TrpE::CmR); SYN2028: E. coli Nissle comprising a deletion in Trp:E and expressing kynureninase from Pseudomonas fluorescens under the control of a constitutive promoter (the synthetic J23119 promoter) integrated into the genome at the HA3/4 site (HA3/4::PSynJ23119-pKYNase KanR TrpE::CmR); SYN2027-R1: a first evolved strain resulting from ALE, derived from the parental SYN2027 strain (Plpp- pKYNase KanR TrpE::CmR EVOLVED STRAIN Replicate 1). SYN2027-R2: a second evolved strain resulting from ALE, derived from the parental SYN2027 strain (Plpp- pKYNase KanR TrpE::CmR EVOLVED STRAIN Replicate 2). SYN2028-R1: a first evolved strain resulting from ALE, derived from the parental SYN2028 strain

(HA3/4::PSynJ23119-pKYNase KanR TrpE::CmR EVOLVED STRAIN Replicate 1). SYN2028-R2: a second evolved strain resulting from ALE, derived from the parental SYN2028 strain (HA3/4::PSynJ23119-pKYNase KanR TrpE::CmR EVOLVED STRAIN Replicate 1).

[034] FIG. 14A and FIG. 14B depict dot plots showing intratumoral kynurenine depletion by strains producing kynureninase from Pseudomonas

fluorescens. FIG. 14A depicts a dot plot showing a intra tumor concentrations observed for the kynurenine consuming strain SYN1704, carrying a constitutively expressed Pseudomonase fluorescens kynureninase on a medium copy plasmid. FIG. 14B. depicts a dot plot showing a intra tumor concentrations observed for the kynurenine consuming strain SYN2028 carrying a constituively expressed chromosomally integrated copy of Pseudomonase fluorescens kynureninase. The IDO inhibitor INCB024360 is used as a positive control.

[035] FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D depict dot plots showing concentrations of intratumoral kynurenine (FIG. 15A) intratumoral tryptophan (FIG. 15B), plasma kynurenine (FIG. 15C) and plasma tryptophan (FIG. 15D) measured in mice administered either saline, or SYN1704. A significant reduction in intratumoral (P<0.001) and plasma (P<0.005) concentration of kynurenine was observed for the kynurenine consuming strain SYN1704 compared to saline control, while tryptophan levels remained constant.

[036] FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G, and FIG. 16H depict schematics of non-limiting examples of embodiments, of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included. FIG. 16A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for Tryptophan decarboxylase, e.g., from Catharanthus roseus, which converts tryptophan to tryptamine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 16B depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate

aminotransferase, e.g., from E. coli, or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole- 3 -acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 16C depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan.

Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus or tdc from Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli), which converts tryptophan to indole-3- acetaldehyde and FICZ, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 16D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for cyp79B2,

(tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3

(tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), which together convert tryptophan to indole-3-acetonitrile, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 16E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising

ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan 2,3- dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine— oxoglutarate transaminase, e.g., from S. cerevisae) which together convert tryptophan to kynurenine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 16F depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising

ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan 2,3- dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine— oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens or AADAT

(Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB 1 (Kynurenine— oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine— oxoglutarate transaminase 3, e.g., from homo sapiens, which together produce kynureninic acid from tryptophan, under the control of an inducible promoter, e.g., an FNR promoter. FIG. 16G depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for tnaA (tryptophanase, e.g., from E. coli), which converts tryptophan to indole, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 16H depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. The genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g., from

Arabidopsis thaliana) under the control of an inducible promoter, e.g. an FNR promoter. The engineered bacterium shown in any of FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G and FIG. 16H may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.

[037] FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, and FIG. 17E depict schematics of exemplary embodiments, of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In FIG. 17A, the optional circuits for tryptophan production are as depicted and described in FIG. 1A. The strain optionally comprises additional circuits as depicted and/or described in FIG. IB and/or FIG. 1C and/or FIG. ID. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 ( L-tryptophan aminotransferase, e.g., from S.

cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or taal (L- tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L- tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) and iadl ( Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAOl (Indole- 3 -acetaldehyde oxidase, e.g., from Arabidopsis thaliana) which together produce indole- 3 -acetic acid from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 17B the optional circuits for tryptophan production are as depicted and described in FIG. 1A. The strain optionally comprises additional circuits as depicted and/or described in FIG. IB and/or FIG. 1C and/or FIG. ID. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., tdc from Catharanthus roseus or tdc from Clostridium sporogenes) ot tynA (Monoamine oxidase, e.g., from E. coli) and or iadl (Indole- 3 -acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAOl (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 17C the optional circuits for tryptophan production are as depicted and described in FIG. 1A. The strain optionally comprises additional circuits as depicted and/or described in FIG. IB and/or FIG. 1C and/or FIG. ID. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L- tryptophan oxidase, e.g., from streptomyces sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 ( indole-3- pyruvate monoxygenase, e.g., from Arabidopsis thaliana) e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 17D the optional circuits for tryptophan production are as depicted and described in FIG. 1A. The strain optionally comprises additional circuits as depicted and/or described in FIG. IB and/or FIG. 1C and/or FIG. ID. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 17E the optional circuits for tryptophan production are as depicted and described in FIG. 1A. The strain optionally comprises additional circuits as depicted and/or described in FIG. IB and/or FIG. 1C and/or FIG. ID. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana and cyp71al3 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nitl (Nitrilase, e.g., from Arabidopsis thaliana) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter, the engineered bacterium shown in any of FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, and FIG. 17E may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.

[038] FIG. 18A and FIG. 18B depict schematics of cicuits for the production of indole metabolites. FIG. 18A depicts a schematic of an indole-3-propionic acid (IP A) synthesis circuit. IPA produced by the gut microbiota has a significant positive effect on barrier integrity. IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate

Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and To 11- like Receptor 4; Immunity 41, 296-310, August 21, 2014, and US Patent Publication No.

20150258151). In some embodiments, IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax

benzoatilyticus) and indole-3-acrylate reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole- 3 -acrylic acid, and indole-3- acrylate reductase converts indole- 3 -acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. FIG. 18B depicts a schematic of another indole-3-propionic acid (IP A) synthesis circuit. Enzymes are as follows: 1. TrpDH: tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108; FldHl/FldH2: indole- 3 -lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole- 3 -lactate

dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; Acul: acrylyl-CoA reductase, e.g., from

Rhodobacter sphaeroides. Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, AD(P) and water to (indol~3-yl)pyruvate, N¾, NAD(P)H and H⁺. Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indol- 3yl)pyruvate and NADH and H+ to indole- 3 -lactate and NAD+. Indole-3-propionyl- CoA:indole-3-lactate CoA transferase (FldA) converts indole-3-lactate and indol-3- propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA. Indole-3-acrylyl- CoA reductase (FldD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole- 3 -lactate dehydratase (FldBC) converts indole-3- lactate-CoA to indole-3-acrylyl-CoA. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan.

[039] FIG. 19A and FIG. 19B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production. FIG. 19A depicts a schematic showing intermediates in tryptophan biosynthesis and the gene products catalyzing the production of these intermediates. Phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) are used to generate 3-deoxy-D-arabino- heptulosonate 7-phosphate (DAHP). DHAP is catabolized to chorismate and then anthranilate, which is converted to tryptophan (Trp) by the tryptophan operon.

Alternatively, chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe). In the serine biosynthesis pathway, D-3-phosphoglycerate is converted to serine, which can also be a source for tryptophan biosynthesis. AroG, AroF, AroH: DAHP synthase catalyzes an aldol reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). There are three isozymes of DAHP synthase, each specifically feedback regulated by tyrosine (AroF), phenylalanine (AroG) or tryptophane AroH). roB;

Dehydroquinate synthase (DHQ synthase) is involved in the second step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids, DHQ synthase catalyzes the eyciization of 3-deoxy-D-arabino-heptu!osomc acid 7 -phosphate (DAHP) to dehydroquinate (DHQ). AroD: 3- Dehydroquinate dehydratase (DHQ dehydratase) is involved in the 3rd step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ dehydratase catalyzes the conversion of DHQ to 3-dehydroshikimate and introduces the first double bond of the aromatic ring. AroE, YdiB: E. coli expresses two shikimate dehydrogenase paralogs, AroE and YdiB. Shikimate dehydrogenase is involved in the 4th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme converts 3-dehydroshikimate to shikimate by catalyzing the NADPH linked reduction of 3-dehydro-shikimate. AroL/AroK: Shikimate kinase is involved in the fifth step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. Shikimate kinase catalyzes the formation of shikimate 3- phosphate from shikimate and ATP. There are two shikimate kinase enzymes, I (AroK) and II (AroL). AroA: 3 -Phospho shikimate- 1-carboxyvinyltransferase (EPSP synthase) is involved in the 6th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. EPSP synthase catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to the hydroxyl group of carbon 5 of shikimate 3-phosphate with the elimination of phosphate to produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP). AroC: Chorismate synthase (AroC) is involved in the 7th and last step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point compound that serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis. This reaction introduces a second double bond into the aromatic ring system. TrpEDCAB (E coli trp operon): TrpE (anthraniiate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L-glutamate. Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5'-phosphoribosyl)-anthranilate. The phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein. Bifunctional phosphoribosylanthranilate isomerase / indole-3-glycerol phosphate synthase (TrpC) carries out the third and fourth steps in the tryptophan biosynthesis pathway. The phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori rearrangement of its substrate into

carboxyphenylaminodeoxyribulose phosphate. The indole-glycerol phosphate synthase activity of TrpC catalyzes the ring closure of this product to yield indole-3-glycerol phosphate. The TrpA polypeptide (TSase a) functions as the a subunit of the tetrameric (α2-β2) tryptophan synthase complex. The TrpB polypeptide functions as the β subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the β reaction. TnaA: Tryptophanase or tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the cleavage of L- tryptophan to indole, pyruvate and NH4+. PheA: Bifunctional chorismate mutase / prephenate dehydratase (PheA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis. TyrA: Bifunctional chorismate mutase / prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis. TyrB, ilvE, AspC: Tyrosine

aminotransferase (TyrB), also known as aromatic- amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis. TyrB catalyzes the transamination of 2-ketoisocaproate, p- hydroxyphenylpyruvate, and phenylpyruvate to yield leucine, tyrosine, and

phenylalanine, respectively. TyrB overlaps with the catalytic activities of branched- chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine. SerA: D- 3-phosphoglycerate dehydrogenase catalyzes the first committed step in the

biosynthesis of L-serine. SerC: The serC-encoded enzyme,

phosphoserine/phosphohydroxythreonine aminotransferase, functions in the biosythesis of both serine and pyridoxine, by using different substrates. Pyridoxal 5'-phosphate is a cofactor for both enzyme activities. SerB: Phosphoserine phosphatase catalyzes the last step in serine biosynthesis. Steps which are negatively regulated by the Trp Repressor (2), Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or positively regulated by trptophan (6) are indicated. FIG. 19B depicts a schematic showing exemplary engineering strategies which can improve tryptophan production. Each of these exemplary strategies can be used alone or two or more strategies can be combined to increase tryptophan production. Intervention points are in bold, italics and underlined. In one embodiment of the disclosure, bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEfbr, and/or TrpD, and/or TrpC, and/or Trp A, and/or TrpB. In one embodiment, endogenous TnaA is knocked out through mutation(s) and/or deletion(s). In one embodiment, bacteria are engineered to comprise one or more additional copies of SerA. In one embodiment, bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter. In one embodiment, endogenous PheA is knocked out through mutation(s) and/or deletion(s). In one embodiment, bacteria are engineered to comprise a circuit for the expression of kynureninase, e.g., kynureninase from Pseudomonas fluorescens or human kynureninase, Without wishing to be bound by theory, addition of a circuit expressing kynureninase will increase production of tryptophan if kynurenine is present in the extracellular environment, such as for example a tumor microenvironment. A strain comprising circuitry to enhance tryptophan production and circuitry for the consumption of kynurenine reduces kynurenine levels while increasing tryptophan levels, e.g., in the extracellular environment, such as a tumor microenvironment, thereby more effectively changing the tryptophan to kynurenine ratio. In one embodiment, two or more of the strategies depicted in the schematic of FIG. 19B are engineered into a bacterial strain.

Alternatively, other gene products in this pathway may be mutated or overexpressed.

[040] FIG. 20A, FIG. 20B, and FIG. 20C depict schematics of exemplary embodiments, of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan and the degradation of kynurenine. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 1A and/or and/or FIG. IB, and/or FIG. 1C, and/or FIG. ID for the production of tryptophan. In one embodiment, the tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. Optionally, Trp Repressor and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. Additionally, AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production, and the strain further optionally comprises either a wild type or a feedback resistant serA gene. The bacteria may also optionally include gene sequence(s) for the expression of YddG to assist in tryptophan export. Additionally, the bacteria further comprise kynureninase, e.g., kynureninase from Pseudomonas fluorescens. When extracellular kynurenine is present, it is imported into the cell and is then converted by kynureninase into anthranilate. Anthranilate is then metabolized into tryptophan via the TrpDCAB pathway enzymes, resulting in further increased levels of tryptophan production.

[041] FIG. 21A, FIG. 21B, and FIG. 21C depict schematics of exemplary embodiments, of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). FIG. 21A a depicts non- limiting example of a tryptamine producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 1A and/or FIG. IB and/or FIG. 1C and/or FIG. ID. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus or tdc from Clostridium sporogenes), which converts tryptophan into tryptamine. FIG. 21B depicts a non-limiting example of an indole- 3 -acetate producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 1A and/or FIG. IB and/or FIG. IC and/or FIG. ID. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole- 3 -acetaldehyde into indole-3-acetate. FIG. 21C depicts a non-limiting example of an indole-3-propionate- producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 1A and/or FIG. IB and/or FIG. IC and/or FIG. ID. Additionally, the strain comprises a circuit as described in FIG. 18B, comprising trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indol-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole- 3 -lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or Acul: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldHl and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole- 3 -lactate).

[042] FIG.22A, FIG. 22B, FIG. 22C, and FIG. 22D depict bar graphs showing tryptophan production by various engineered bacterial strains. FIG.22A depicts a bar graph showing tryptophan production by various tryptophan producing strains. The data show expressing a feedback resistant form of AroG (AroG fbr ) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE

(trpE fbr ) has a positive effect on tryptophan production. FIG. 22B shows tryptophan production from a strain comprising a tet-trpE fbr DCBA, tet-aroG fb construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes E. coli two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically). FIG. 22C depicts a bar graph showing improved tryptophan production by engineered strain comprising AtrpRAtnaA, tet-trpE^DCBA, tet-aro '^r through the addition of serine. FIG. 22D depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476.

SYN2126 AtrpRAtnaA. AtrpRAtnaA, tet-aroGfbr. SYN2339 comprises AtrpRAtnaA, tet-aroGfbr, tet-trpEfbrDCBA. SYN2473 comprises AtrpRAtnaA, tet-aroGfbr-serA, tet- trpEfbrDCBA. SYN2476 comprises AtrpRAtnaA, tet-trpEfbrDCBA. Results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.

[043] FIG. 23B depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342. SYN2126: comprises AtrpR and AtnaA (AtrpRAtnaA). SYN2339 comprises circuitry for the production of tryptophan (AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr (pl5A)). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iadl incorporated at the end of the second construct (AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr-trpDH- ipdC-iadl (pl5A)). SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 converts all trypophan it produces into IAA.

[044] FIG. 23C depicts a bar graph showing tryptophan and tryptamine production for strains SYN2339, SYN2340, and SYN2794. SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine and comprises AtrpRAtnaA, tetR-P_tet-trpE^DCBA (pSClOl), tetR-P_tet-aroG*¹ (pl5A). SYN2340 comprises AtrpRAtnaA,

(pl5A). SYN2794 comprises AtrpRAtnaA, tetR-P_tet-trpE^DCBA (pSClOl), tetR-P_tet-aroG*¹- tdccs (pl5A). Results indicate that Tdcc_s from Clostridium sporogenes is more efficient the Tdcc_r from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine. [045] FIG. 24 depicts a map of integration sites within the E. coli Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites.

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

[047] FIG. 26 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).

[048] FIG. 27 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellular ly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[049] FIG. 28 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.

[050] FIG. 29 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP-binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[051] FIG. 30 depicts a schematic of the outer and inner membranes of a gram- negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the extracellular space, e.g., therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.

[052] FIG. 31 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide

(hexagons).

[053] FIG. 32A, FIG. 32B, and FIG. 32C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide of interest, such as, IDO, TDO or any other tryptophan synthesis, indole synthesis or catabolism enzyme described herein, is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (FIG. 32A and FIG. 32B) or a tet-inducible promoter (FIG. 32C). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g. , FNR-inducible promoter), promoters induced by IBD or tumor specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose is used. In some embodiments, expression of thereaputic peptide is induced by a tumor specific metabolite. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct, as shown in FIG. 32B and FIG. 32C.

[054] FIG. 32D and FIG. 31E depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, such as, IDO, TDO or any other tryptophan synthesis, indole synthesis or catabolism enzyme, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpl. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a tet promoter (FIG. 32D) or an inducible promoter, such as oxygen level-dependent promoters (e.g. , FNR- inducible promoter, FIG. 31E), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. In some embodiments, expression of therapeutic peptide is induced by a tumor specific metabolite.

[055] FIG. 33A, FIG. 33B, and FIG. 33C depict schematics of other non- limiting embodiments, of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (ParaBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. FIG. 33A also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell. FIG. 33B depicts a non- limiting

embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit. FIG. 33C depicts another non- limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.

[056] FIG. 34 depicts one non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[057] FIG. 35 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.

[058] FIG. 36 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death.

[059] FIG. 37 depicts one non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the

recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[060] FIG. 38 depicts a one non-limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce a equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti- toxin decays completely, and the cell dies.

[061] FIG. 39 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al., "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synthetic Biology (2015) 4: 307-316.

[062] FIG. 40 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown in Table 4 (Pfnrl-5). Different FNR-responsive promoters were used to create a library of anaerobic-inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+0₂) or anaerobic conditions (-0₂). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β- galactosidase colorimetric assays.

[063] FIG. 41A, FIG. 41B and FIG. 41C depict schematic representations of the lacZ gene under the control of an exemplary FNR promoter (P^_rs) and

corresponding graphical data. FIG. 41A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P_fnrs)- LacZ encodes the β- galactosidase enzyme and is a common reporter gene in bacteria. FIG. 41B depicts FNR promoter activity as a function of β-galactosidase activity in SYN340. SYN340, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr under anaerobic conditions. FIG. 41C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

[064] FIG. 42A , FIG. 42B, FIG. 42C, and FIG. 42D depict bar graphs, schematic, and dot blot, respectively, showing the structure or activity of reporter constructs. FIG. 42A and FIG. 42B depict bar graphs of reporter constructs activity. FIG. 69A depicts a graph of an ATC-inducible reporter construct expression and FIG. 42B depicts a graph of a nitric oxide-inducible reporter construct expression. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible P_tet-GFP reporter construct or the nitric oxide inducible P_nsrR-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units. FIG. 42C depicts a schematic of the constructs. FIG. 42D depicts a dot blot of bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR- inducible promoter. DSS-treated mice serve as exemplary models for HE. As in HE subjects, the guts of mice are damaged by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice.

[065] FIG. 43A depicts a graph showing bacterial cell growth of a Nissle thyA auxotroph strain (thyA knock-out) in various concentrations of thymidine. A

chloramphenicol-resistant Nissle thyA auxotroph strain was grown overnight in LB + lOmM thymidine at 37C. The next day, cells were diluted 1 : 100 in 1 mL LB + lOmM thymidine, and incubated at 37C for 4 hours. The cells were then diluted 1 : 100 in 1 mL LB + varying concentrations of thymidine in triplicate in a 96-well plate. The plate is incubated at 37C with shaking, and the OD600 is measured every 5 minutes for 720 minutes. This data shows that Nissle thyA auxotroph does not grow in environments lacking thymidine. FIG. 43B depicts a bar graph of Nissle residence in vivo of wildtype Nissle versus Nissle thyA auxotroph (thyA knock-out). Streptomycin- resistant Nissle (wildtype or thyA auxotroph) was administered to mice via oral gavage without antibiotic pre- treatment. Fecal pellets from 6 total mice were monitored post- administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. Each bar represents the number of Nissle recovered from the fecal samples each day for 7 consecutive days. There were no bacteria recovered in fecal samples from mice gavaged with Nissle thyA auxotroph bacteria after day 3. This data shows that the Nissle thyA auxotroph does not persist in vivo in mice.

[066] FIG. 44 depicts a graph of Nissle residence in vivo. Streptomycin- resistant Nissle was administered to mice via oral gavage without antibiotic pre- treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse

gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

[067] FIG. 45 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each

compartment were determined by serial dilution plating.

[068] FIG. 46A, FIG. 46B, FIG. 46C, FIG. 46D, FIG. 46E, and FIG. 46F depict schematics of exemplary bacteria of the disclosure. The bacteria comprise one or more gene(s) or gene sequence(s) which are optionally expressed from an inducible promoter, e.g., a FNR- inducible promoter. The bacteria may also include an

auxotrophy, e.g., deletion of thyA (Δ thyA). Non-limiting examples of bacterial strains are listed. FIG. 46A shows a schematic depicting an exemplary bacterium having a non- native secretion system used to secrete a therapeutic peptide (kynureninase).

Alternatively or additionally, kynureninase may also optionally be expressed in the bacteria but not secreted to allow for the bacterium to consume and degrade kynurenine. The bacterium is further capable of producing tryptophan. Optionally, the bacterium may also comprise one or more of the mutations/deletions depicted or described in FIG. 19A and FIG. 19B or elsewhere herein. Secretion system refers to a native or non- native secretion mechanism capable of secreting the anti-cancer molecule from the bacterial cytoplasm. Non- limiting examples of secretion systems for gram negative bacteria include the type III, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. FIG. 46B depicts a schematic showing a bacterium comprising an exemplary kynurenine degradation circuit. Kynurenine is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynureninase is expressed to metabolize kynurenine to anthranilic acid in the cell. Anthranilic acid may be used for the production of tryptophan. FIG. 46C depicts a schematic of a bacterium comprising one or more gene(s) or gene sequence(s) for the expression of the essential gene tnaB, 5- methyltetrahydrofolate-homocysteine methyltransferase {mtr), tryptophan transporter, and the enzymes IDO and TDO to convert tryptophan into kynurenine. FIG. 46D depicts a schematic of a bacterium comprising one or more gene(s) or gene sequence(s) for the expression of one or more kynurenine biosynthetic enzyme(s), e.g., IDO and/or TDO and/or one or more enzymes depicted or described in FIG. 16E and/or FIG. 16F (kynurenic acid production) or elsewhere herein. The bacterium further comprises one or more gene(s) or gene sequence(s) for the expression of one or more tryptophan biosynthetic enzyme(s), e.g., as depicted and described in FIG. 1A and/or FIG. IB and/or FIG. 1C and/or FIG. ID or as described elsewhere herein, and optionally one or more gene(s) or gene sequence(s) for the expression of AroP. Optionally, the bacterium may also comprise one or more of the mutations/deletions depicted or described in FIG. 19B or elsewhere herein. FIG. 46E depicts a schematic of a bacterium comprising one or more gene(s) or gene sequence(s) for the expression of one or more tryptophan biosynthetic enzyme(s), e.g., as depicted and described in FIG. 1A and/or FIG. IB and/or FIG. 1C and/or FIG. ID or as described elsewhere herein. Optionally, the bacterium may also comprise one or more of the mutations/deletions depicted or described in FIG. 19B or elsewhere herein. The bacterium also comprises one or more gene(s) or gene sequence(s) for the expression of one or more enzyme(s) for the production of one or more indole(s), e.g., as depicted and described in FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17D, FIG. 17E, FIG. 17E, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16G, FIG. 16H, FIG. 18A, and FIG. 18B, and/or elsewhere herein. The bacterium optionally comprises one or more gene(s) or gene sequence(s) for the expression of AroP. FIG. 46F depicts a schematic of a bacterium comprising one or more gene(s) or gene sequence(s) for the expression of one or more tryptophan biosynthetic enzyme(s), e.g., as depicted and described in FIG. 1A and/or FIG. IB and/or FIG. 1C and/or FIG. ID or as described elsewhere herein. Optionally, the bacterium may also comprise one or more of the mutations/deletions depicted or described in FIG. 19B or elsewhere herein. The bacterium also comprises one or more gene(s) or gene sequence(s) for the expression of one or more enzyme(s) for the production of tryptamine, e.g., as depicted and described in FIG. 16A and/or elsewhere herein. The bacterium optionally comprises one or more gene(s) or gene sequence(s) for the expression of AroP.

[069] FIG. 47 depicts a schematic of a polypeptide of interest displayed on the surface of the bacterium. A non-limiting example of such a therapeutic protein is a scFv. The polypeptide is expressed as a fusion protein, which comprises a outer membrane anchor from another protein, which was developed as part of a display system. Non- limiting examples of such anchors are described herein and include LppOmpA,

NGIgAsig-NGIgAP, InaQ, Intimin, Invasin, pelB-PAL, and blcA/BAN. In a

nonlimiting example a bacterial strain which has one or more diffusible outer membrane phenotype ("leaky membrane") mutation, e.g. , as described herein.

[070] Figs. 48A-48D depict schematics of non-limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (Fig. 48A and Fig. 48B), which also contains a chromosomal component (shown in Fig. 48C and Fig. 48D). The Biosafety Plasmid System Vector comprises Kid Toxin and R6K minimal ori, dapA (Fig. 48A) and thyA (Fig. 48B) and promoter elements driving expression of these components. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, in which a first protein of interest (POI1) and/or a second protein of interest, e.g., a transporter (POI2), and/or a third protein of interest (POI3) are expressed from an inducible or constitutive promoter. Fig. 48C and Fig. 48D depict schematics of the gene organization of the chromosomal component of a biosafety system. Fig. 48C depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter. Fig. 48D depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter. If the plasmid containing the functional DapA is used (as shown in Fig. 48A), then the chromosomal constructs shown in Fig. 48C and Fig. 48D are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in Fig. 48B), then the chromosomal constructs shown in Fig. 48C and Fig. 48D are knocked into the ThyA locus. In this system, the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.

[071] Fig. 49 depicts the gene organization of exemplary construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.

[072] Fig. 50A depicts a "Oxygen bypass switch" useful for aerobic pre- induction of a strain comprising one or proteins of interest (POI), e.g., one or more anticancer molecules or immune modulatory effectors (POI1) and a second set of one or more proteins of interest (POI2), e.g., one or more transporter(s)/importer(s) and/or exporter(s), under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active effector molecules prior to administration. This can be done by pre-inducing the expression of these effectors as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more effectors or proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more effectors or proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites.

[073] FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ, The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system,

FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo. In some embodiments, a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

[074] Fig. 50B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y. By using a ribosome binding site optimization strategy, the levels of Fnr expression can be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bio informatics tools for optimization of RBS are known in the art.

[075] Fig. 50C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bio informatics tools for optimization of RBS are known in the art. In one strategy, arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of Pfnrs-POI constructs are maintained to allow for strong in vivo induction.

[076] Fig. 51 depicts the gene organization of an exemplary construct, comprising a cloned POI gene under the control of a Tet promoter sequence and a Tet repressor gene.

[077] Fig. 52 depicts the gene organization of an exemplary construct comprising Lacl in reverse orientation, and a IPTG inducible promoter driving the expression of one or more POIs. In some embodiments, this construct is useful for pre- induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.

[078] In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a bio safety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some

embodiments, the construct is used in combination with construct expressing a second POI, e.g., a transporter, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be

constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is located on a plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct is employed in a biosafety system, such as the system shown in Fig. 48A, Fig. 48B, Fig. 48C, and Fig. 48D. In some

embodiments, the construct is integrated into the genome at one or more locations described herein.

[079] Fig. 53A, Fig. 53B, and Fig. 53C depict schematics of non-limiting examples of constructs constructs for the expression of proteins of interest POI(s). Fig 53A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, this construct is used alone. In some embodiments, the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of a POIl and/or a POI2 prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POI3 expression construct.

[080] In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).

[081] Fig. 53B depicts a schematic of a non- limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter. For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multicopy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre- induction and pre-loading of POI and/or POI2 and/or POI3 prior to in vivo

administration. In a non- limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POD expression construct. [082] Fig. 53C depicts a schematic of a non-limiting example of the organization of a construct for the expression of protein(s) of interest POI(s) under the control of an arabinose inducible promoter. The arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and POI. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre- induction and pre-loading of POI 1 and/or POI2 and/or POI3 prior to in vivo

administration. In a non- limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POI3 expression construct.

[083] Fig. 54A depicts a schematic of the gene organization of a PssB promoter. The ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair. The PssB promoter was cloned in front of a LacZ reporter and beta-galactosidase activity was measured.

[084] Fig. 54B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1: 100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g. , the gut, but prevent survival under aerobic conditions (biosafety switch).

[085] FIG. 455A and FIG. 55B depict a schematic diagrams of a wild-type clbA construct (FIG. 46A) and a schematic diagram of a clbA knockout construct (FIG. 55B).

[086] FIG. 56 depicts a schematic of a design-build-test cycle. Steps are as follows: 1 : Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animla disease model; 9. Assimilate into the microbiome; 10.

Develop understanding of in vivo PK and dosing regimen.

[087] FIG. 57 depicts a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. Step 1 depicts the parameters for starter culture 1 (SCI): loop full - glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm. Step 2 depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SCI, duration 1.5 hours, temperature 37° C, shaking at 250 rpm. Step 3 depicts the parameters for the production bioreactor: inoculum - SC2, temperature 37° C, pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300- 1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours. Step 4 depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re- suspension 10% glycerol/PBS. Step 5 depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.

Description of Embodiments

[088] The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating or treating disorders associated with tryptophan metabolism. The genetically engineered bacteria are capable of modulating tryptophan metabolism along the serotonin and kynurenine pathways. In

someembodiments, the genetically engineered bacteria are capable of modulating tryptophan metabolism along the serotonin and kynurenine pathways, under certain environmental conditions, such as those in the mammalian gut and/or the tumor microenvironment. In modulating tryptophan levels and its metabolites, in some embodiments, the genetically engineered bacteria are responding to the environmental inflammatory status (pro-inflammatory or anti-inflammatory/immunosuppressive) or oxygen status (high or low oxygen status) with the production of one or more gene products (i.e. through inducible promoters). In some embodiments, the genetically engineered bacteria modulate the ratio of trypophan metabolites in serum, e.g. in the circulation systemically or locally, i.e. , the bacteria may for example modulate the TRP:KYN ratio or the KYNA:QUIN ratio in certain environmental settings. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s), the gene products of which are able to influence or modulate the

inflammatory status of the environment. In some embodiments, the genetically engineered bacteria produce kynurenine and/or kynurenine metabolites, resulting in a more immuno-suppressive environment. In some embodiments, the genetically engineered bacteria produce tyrptophan, resulting in a more pro-inflammatory environment.

[089] In some embodiments, the genetically engineered bacteria reduce inflammation through modulation of the kynurenine pathway and can also perform one more more additional functions. In some embodiments, the genetically engineered bacteria reduce inflammation through modulation of the kynurenine pathway and also reduce one or more other toxic substances. In some embodiments, the genetically engineered bacteria reduce inflammation through modulation of the kynurenine pathway and can also perform one more more additional functions. In some

embodiments, the genetically engineered bacteria reduce inflammation through modulation of the kynurenine pathway and additionally produce one or molecules that improve gut barrier function, Non-limiting examples of such molecules include one or more short chain fatty acids, e.g. , butyrate, propionate, and/or acetate. In some embodiments, the engineered bacteria reduce inflammation through modulation of the kynurenine pathway and produce GLP-2. In some embodiments, the engineered bacteria reduce inflammation through modulation of the kynurenine pathway and produce SOD. In some embodiments, the engineered bacteria reduce inflammation through modulation of the kynurenine pathway and produce an ant i- inflammatory cytokine, such as IL- 10. In embodiments, of the disclosure, the genetically engineered bacteria which reduce inflammation through modulation of the kynurenine pathway also produce one or more of (1) anti- inflammatory interleukins, (2) Superoxide Dismutaase (SOD), (3) GLP-2.

[090] In some embodiments, the genetically engineered bacteria increase inflammation through modulation of the kynurenine pathway and/or tryptophan production and can also perform one more more additional functions. In some embodiments, the genetically engineered bacteria increase inflammation through modulation of the kynurenine pathway and/or tryptophan production and also produce inflammatory cytokines and/or other immune stimulatory molecules, including but not limited to e.g., IL- 12, IL-2, IL- 15, IL- 18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In some embodiments, the genetically engineered bacteria increase inflammation through modulation of the kynurenine pathway and/or tryptophan production and can also produce a checkpoint inhibitor, including but not limited to antibodies and/or single chain antibodies directed against CTLA-4, PD1, and/or PDL1. Other checkpoint inhibitors are known in the art. In some embodiments, the genetically engineered bacteria reduce inflammation through modulation of the kynurenine pathway and/or tryptophan production and can also produce lytic peptides and other cytotoxic peptide, as described in co-owned US Provisional Application 62/335,940, the contents of which is herein incorporated by reference in its entirety. In some embodiments, the genetically engineered bacteria increase inflammation through modulation of the kynurenine pathway and/or tryptophan production and can also produce another metabolic modulator, including but not limited to arginase. In some embodiments, the genetically engineered bacteria increase inflammation through modulation of the kynurenine pathway and/or tryptophan production also produce one or more of (1) Inflammatory cytokines, (2) checkpoint inhibitors, (3) lytic peptides and/or other cytotoxic peptides, and (4) metabolic modulators.

[091] In other embodiments, the genetically engineered bacteria are capable of modulating tryptophan metabolism along the serotonin and kynurenine pathways and produce one or more other effector molecules, such as any of th effector molecules described herein.

[092] In any of the described embodiments, the engineered bacteria may further comprise one or more of more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g. , thyA auxo trophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

[093] In some embodiments, any one or more of the payload or therapeutic circuits (e.g. , tryp metabolism modulating circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by a constitutive promoter. In some embodiments, any one or more of the payload or therapeutic circuits (e.g. , e.g., tryp metabolism modulating circuits ) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by a tissue- specific promoter. In some

embodiments, any one or more of the payload or therapeutic circuits (e.g. , tryp metabolism modulating circuits ) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by an inducible promoter. In some embodiments, any one or more of the payload or therapeutic circuits (e.g., tryp metabolism modulating circuits) and/or any one or more of the additional circuits (e.g. , auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by an inducible promoter that is responsive to environmental conditions, factors, or cues, e.g. , environmental conditions, factors, or cues found in the mammalian gut or in the tumor micorenvironment. Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by - specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g. , can be exogenously added) in the gut, e.g., arabinose and tetracycline.

[094] In some embodiments, any one or more of the payload or therapeutic circuits (e.g. , tryp metabolism modulating circuits ) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be present on one or more low copy or high copy plasmids. In some embodiments, any one or more of the payload or therapeutic circuits (e.g. , tryp metabolism modulating circuits ) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be integrated into the bacterial chromosome.

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

[096] "Intratumoral administration" is meant to include any and all means for microorganism delivery to the intratumoral site and is not limited to intratumoral injection means. Examples of delivery means for the engineered microrganisms is discussed in detail herein.

[097] "Cancer" or "cancerous" is used to refer to a physiological condition that is characterized by unregulated cell growth. In some embodiments, cancer refers to a tumor. "Tumor" is used to refer to any neoplastic cell growth or proliferation or any pre-cancerous or cancerous cell or tissue. A tumor may be malignant or benign. Types of cancer include, but are not limited to, adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma tumors, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytomas, brain stem glioma, craniopharyngioma, ependymoma), bronchial tumors, central nervous system tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, heart cancer, Kaposi sarcoma, kidney cancer, largyngeal cancer, hypopharyngeal cancer, leukemia (e.g. , acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia), liver cancer, lung cancer, lymphoma (e.g. , AIDS- related lymphoma, Burkitt lymphoma, cutaneous T cell lymphoma, Hodgkin lymphoma, Non-Hodgkin lymphoma, primary central nervous system lymphoma), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer (e.g. , basal cell carcinoma, melanoma), small intestine cancer, stomach cancer, teratoid tumor, testicular cancer, throat cancer, thymus cancer, thyroid cancer, unusual childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macrogloblulinemia, and Wilms tumor. Side effects of cancer treatment may include, but are not limited to, opportunistic autoimmune disorder(s), systemic toxicity, anemia, loss of appetite, irritation of bladder lining, bleeding and bruising (thrombocytopenia), changes in taste or smell, constipation, diarrhea, dry mouth, dysphagia, edema, fatigue, hair loss (alopecia), infection, infertility, lymphedema, mouth sores, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infections, and/or problems with memory and concentration (National Cancer Institute).

[098] "Hypoxia" is used to refer to reduced oxygen supply to a tissue as compared to physiological levels, thereby creating an oxygen-deficient environment. "Normoxia" refers to a physiological level of oxygen supply to a tissue. Hypoxia is a hallmark of solid tumors and characterized by regions of low oxygen and necrosis due to insufficient perfusion (Groot et ah, 2007).

[099] As used herein, "anti-inflammation molecules" and/or "gut barrier function enhancer molecules" include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), kynurenine, GLP-2, GLP- 1, IL- 10, IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, PGD₂, and kynurenic acid, as well as other molecules disclosed herein. Such molecules may also include compounds that inhibit pro-inflammatory molecules, e.g. , a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-γ, IL- Ιβ, IL-6, IL-8, IL- 17, and/or chemokines, e.g. , CXCL-8 and CCL2. A molecule may be primarily anti- inflammatory, e.g., IL- 10, or primarily gut barrier function enhancing, e.g., GLP-2. A molecule may be both anti- inflammatory and gut barrier function enhancing. An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g. , elafin is encoded by the PI3 gene. Alternatively, an anti- inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g. , butyrate. These molecules may also be referred to as therapeutic molecules.

[0100] As used herein, "diseases and conditions associated with gut

inflammation and/or compromised gut barrier function" include, but are not limited to, inflammatory bowel diseases, diarrheal diseases, and related diseases. "Inflammatory bowel diseases" and "IBD" are used interchangeably herein to refer to a group of diseases associated with gut inflammation, which include, but are not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, and indeterminate colitis. As used herein, "diarrheal diseases" include, but are not limited to, acute watery diarrhea, e.g., cholera; acute bloody diarrhea, e.g., dysentery; and persistent diarrhea. As used herein, related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis,

proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis.

[0101] Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of diarrhea, bloody stool, mouth sores, perianal disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron deficiency, anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal development, inflammation of the skin, inflammation of the eyes, inflammation of the joints, inflammation of the liver, and inflammation of the bile ducts.

[0102] As used herein, "diseases and conditions associated with gut

inflammation and/or compromised gut barrier function" disease or condition associated with gut inflammation and/or compromised gut barrier function may be an a

neurological disorder. As used herein, "neurological disorders" include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing

hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis,

antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg- Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogan' s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier' s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's

encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory

lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatry Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes,

polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome,

postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing

polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis,

undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis.

[0103] Abulia, Agraphia, Alcoholism, Alexia, Alien hand syndrome, Allan- Herndon-Dudley syndrome, Alzheimer's disease, Amaurosis fugax, Amnesia,

Amyotrophic lateral sclerosis (ALS), Aneurysm, Angelman syndrome, Aphasia, Apraxia, Arachnoiditis, Arnold-Chiari malformation, Asperger syndrome, Ataxia, Attention deficit hyperactivity disorder, ATR-16 syndrome, Auditory processing disorder, Autism spectrum, Behcets disease, Bipolar disorder, Bell's palsy, Brachial plexus injury, Brain damage, Brain injury, Brain tumor, Canavan disease, Capgras delusion, Carpal tunnel syndrome, Causalgia, Central pain syndrome, Central pontine myelinolysis, Centronuclear myopathy, Cephalic disorder, Cerebral aneurysm, Cerebral arteriosclerosis, Cerebral atrophy, Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), Cerebral gigantism, Cerebral palsy, Cerebral vasculitis, Cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, Chorea, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic pain, Cockayne syndrome, Coffin- Lowry syndrome, Coma, Complex regional pain syndrome, Compression neuropathy, Congenital facial diplegia, Corticobasal degeneration, Cranial arteritis, Craniosynostosis, Creutzfeldt-Jakob disease, Cumulative trauma disorders, Cushing's syndrome, Cyclothymic disorder, Cytomegalic inclusion body disease (CIBD),

Cytomegalovirus Infection, Dandy- Walker syndrome, Dawson disease, De Morsier's syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, Delayed sleep phase syndrome, Dementia, Dermatomyositis, Developmental coordination disorder, Diabetic neuropathy, Diffuse sclerosis, Diplopia, Disorders of consciousness, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, Dysarthria, Dysautonomia,

Dyscalculia, Dysgraphia, Dyskinesia, Dyslexia, Dystonia, Empty sella syndrome, Encephalitis, Encephalocele, Encephalotrigeminal angiomatosis, Encopresis, Enuresis, Epilepsy, Epilepsy-intellectual disability in females, Erb's palsy, Erythromelalgia, Essential tremor, Exploding head syndrome, Fabry's disease, Fahr's syndrome, Fainting, Familial spastic paralysis, Febrile seizures, Fisher syndrome, Friedreich's ataxia, Fibromyalgia, Foville's syndrome, Fetal alcohol syndrome, Fragile X syndrome, Fragile X-associated tremor/ataxia syndrome (FXTAS), Gaucher's disease, Generalized epilepsy with febrile seizures plus, Gerstmann's syndrome, Giant cell arteritis, Giant cell inclusion disease, Globoid Cell Leukodystrophy, Gray matter heterotopia, Guillain- Barre syndrome, Generalized anxiety disorder, HTLV-1 associated myelopathy, Hallervorden-Spatz syndrome, Head injury, Headache, Hemifacial Spasm, Hereditary Spastic Paraplegia, Heredopathia atactica polyneuritiformis, Herpes zoster oticus, Herpes zoster, Hirayama syndrome, Hirschsprung's disease, Holmes-Adie syndrome, Holoprosencephaly, Huntington's disease, Hydranencephaly, Hydrocephalus,

Hypercortisolism, Hypoxia, Immune-Mediated encephalomyelitis, Inclusion body myositis, Incontinentia pigmenti, Infantile Refsum disease, Infantile spasms,

Inflammatory myopathy, Intracranial cyst, Intracranial hypertension, Isodicentric 15, Joubert syndrome, Karak syndrome, Kearns-Sayre syndrome, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Lateral medullary (Wallenberg) syndrome, Learning disabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Leukodystrophy, Leukoencephalopathy with vanishing white matter, Lewy body dementia, Lissencephaly, Locked-in syndrome, Lou Gehrig's disease (See amyotrophic lateral sclerosis), Lumbar disc disease, Lumbar spinal stenosis, Lyme disease - Neurological Sequelae, Machado-Joseph disease (Spinocerebellar ataxia type 3), Macrencephaly, Macropsia, Mai de debarquement, Megalencephalic leukoencephalopathy with subcortical cysts, Megalencephaly,

Melkersson-Rosenthal syndrome, Menieres disease, Meningitis, Menkes disease, Metachromatic leukodystrophy, Microcephaly, Micropsia, Migraine, Miller Fisher syndrome, Mini-stroke (transient ischemic attack), Misophonia, Mitochondrial myopathy, Mobius syndrome, Monomelic amyotrophy, Motor Neurone Disease - see amyotrophic lateral sclerosis, Motor skills disorder, Moyamoya disease,

Mucopolysaccharidoses, Multi-infarct dementia, Multifocal motor neuropathy, Multiple sclerosis, Multiple system atrophy, Muscular dystrophy, Myalgic encephalomyelitis, Myasthenia gravis, Myelinoclastic diffuse sclerosis, Myoclonic Encephalopathy of infants, Myoclonus, Myopathy, Myotubular myopathy, Myotonia congenita,

Narcolepsy, Neuro-Behget's disease, Neurofibromatosis, Neuroleptic malignant syndrome, Neurological manifestations of AIDS, Neurological sequelae of lupus, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorders,

Neuropathy, Neurosis, Niemann-Pick disease, Non- 24-hour sleep-wake disorder, Nonverbal learning disorder, O'Sullivan-McLeod syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara syndrome, Olivopontocerebellar atrophy, Opsoclonus myoclonus syndrome, Optic neuritis, Orthostatic Hypotension, Otosclerosis, Overuse syndrome, Palinopsia, Paresthesia, Parkinson's disease,

Paramyotonia Congenita, Paraneoplastic diseases, Paroxysmal attacks, Parry-Romberg syndrome, PANDAS, Pelizaeus-Merzbacher disease, Periodic Paralyses, Peripheral neuropathy, Pervasive developmental disorders, Phantom limb / Phantom pain, Photic sneeze reflex, Phytanic acid storage disease, Pick's disease, Pinched nerve, Pituitary tumors, PMG, Polyneuropathy, Polio, Polymicrogyria, Polymyositis, Porencephaly, Post-Polio syndrome, Postherpetic Neuralgia (PHN), Postural Hypotension, Prader- Willi syndrome, Primary Lateral Sclerosis, Prion diseases, Progressive hemifacial atrophy, Progressive multifocal leukoencephalopathy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudotumor cerebri, Quadrantanopia, Quadriplegia, Rabies,

Radiculopathy, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III - see Ramsay-Hunt syndrome, Rasmussen encephalitis, Reflex neurovascular dystrophy, Refsum disease, REM sleep behavior disorder, Repetitive stress injury, Restless legs syndrome, Retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, Rhythmic Movement Disorder, Romberg syndrome, Saint Vitus dance, Sandhoff disease, Schilder's disease (two distinct conditions), Schizencephaly, Sensory processing disorder, Septo-optic dysplasia, Shaken baby syndrome, Shingles, Shy-Drager syndrome, Sjogren's syndrome, Sleep apnea, Sleeping sickness, Snatiation, Sotos syndrome, Spasticity, Spina bifida, Spinal cord injury, Spinal cord tumors, Spinal muscular atrophy, Spinal and bulbar muscular atrophy, Spinocerebellar ataxia, Split- brain, Steele-Richardson-Olszewski syndrome, Stiff-person syndrome, Stroke, Sturge- Weber syndrome, Stuttering, Subacute sclerosing panencephalitis, Subcortical arteriosclerotic encephalopathy, Superficial siderosis, Sydenham's chorea, Syncope, Synesthesia, Syringomyelia, Tarsal tunnel syndrome, Tardive dyskinesia, Tardive dysphrenia, Tarlov cyst, Tay-Sachs disease, Temporal arteritis, Temporal lobe epilepsy, Tetanus, Tethered spinal cord syndrome, Thomsen disease, Thoracic outlet syndrome, Tic Douloureux, Todd's paralysis, Tourette syndrome, Toxic encephalopathy, Transient ischemic attack, Transmissible spongiform encephalopathies, Transverse myelitis, Traumatic brain injury, Tremor, Trichotillomania, Trigeminal neuralgia, Tropical spastic paraparesis, Trypanosomiasis, Tuberous sclerosis, 22ql3 deletion syndrome, Unverricht-Lundborg disease, Vestibular schwannoma (Acoustic neuroma), Von Hippel-Lindau disease (VHL), Viliuisk Encephalomyelitis (VE), Wallenberg's syndrome, West syndrome, Whiplash, Williams syndrome, Wilson's disease,Zellweger syndrome,

[0104] As used herein, "diseases and conditions associated with gut

inflammation and/or compromised gut barrier function" disease or condition associated with gut inflammation and/or compromised gut barrier function may be an a viral or bacterial infection. As used herein, "viral or bacterial infections" include, but are not limited to,Anaplasmosis, Anthrax, Babesiosis, Botulism, Brucellosis, Burkholderia mallei (Glanders), Burkholderia pseudomallei (Melioidosis), Camp ylobacterio sis (Campylobacter), Carbapenem-resistant Enterobacteriaceae (CRE), Chancroid,

Chikungunya, Chlamydia, Ciguatera, Clostridium Difficile Infection, Clostridium Perfringens (Epsilon Toxin), Coccidioidomycosis fungal infection (Valley fever), Creutzfeldt- Jacob Disease, transmissible spongioform (CJD), Crypto sporidio sis (Crypto), Cyclosporiasis, Dengue, 1,2,3,4 (Dengue Fever), Diphtheria, E. Coli infection (E.Coli), Eastern Equine Encephalitis (EEE), Ebola Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Enterovirus Infection, Non-Polio (Non-Polio Enterovirus), Enterovirus Infection, D68 (EV-D68), Giardiasis (Giardia), Gonococcal Infection (Gonorrhea), Granuloma inguinale, Haemophilus Influenza disease, Type B (Hib or H-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes Zoster, zoster VZV (Shingles), Histoplasmosis infection (Histoplasmosis), Human Immunodeficiency Virus/ AIDS (HIV/AIDS), Human Papillomarivus (HPV), Influenza (Flu), Lead

Poisoning, Legionellosis (Legionnaires Disease), Leprosy (Hansens Disease),

Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma venereum infection (LVG), Malaria, Measles, Meningitis, Viral (Meningitis, viral),

Meningococcal Disease, Bacterial (Meningitis, bacterial), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Mumps, Noro virus, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning, Ciguatera), Pediculosis (Lice, Head and Body Lice), Pelvic Inflammatory Disease (PID), Pertussis, Plague; Bubonic, Septicemic, Pneumonic (Plague), Pneumococcal Disease, Poliomyelitis (Polio), Psittacosis, Pthiriasis (Crabs; Pubic Lice Infestation), Pustular Rash diseases (Small pox, monkeypox, cowpox), Q- Fever, Rabies, Ricin Poisoning, Rickettsiosis (Rocky Mountain Spotted Fever), Rubella, Including congenital (German Measles), Salmonellosis gastroenteritis (Salmonella), Scabies Infestation (Scabies), Scombroid, Severe Acute Respiratory Syndrome (SARS), Shigellosis gastroenteritis (Shigella), Smallpox, Staphyloccal Infection, Methicillin- resistant (MRSA), Staphylococcal Food Poisoning, Enterotoxin - B Poisoning (Staph Food Poisoning), Staphylococcal Infection, Vancomycin Intermediate (VISA),

Staphylococcal Infection, Vancomycin Resistant (VRSA), Streptococcal Disease, Group A (invasive) (Strep A), Streptococcal Disease, Group B (Strep-B), Streptococcal Toxic- Shock Syndrome, STSS, Toxic Shock (STSS, TSS), Syphilis, primary, secondary, early latent, late latent, congenital, Tetanus Infection, tetani (Lock Jaw), Trichonosis Infection (Trichinosis), Tuberculosis (TB), Tuberculosis (Latent) (LTBI), Tularemia (Rabbit fever), Typhoid Fever, Group D, Typhus, Vaginosis, bacterial (Yeast Infection), Varicella (Chickenpox), Vibrio cholerae (Cholera), Vibriosis (Vibrio), Viral

Hemorrhagic Fever (Ebola, Lassa, Marburg), West Nile Virus, Yellow Fever, Yersenia (Yersinia), Zika (Zika Virus),

[0105] "Operably linked" refers a nucleic acid sequence, e.g., a gene encoding feedback resistant ArgA, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3 ' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[0106] An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. In some embodiments, the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage. In some embodiments, the metabolites may be gut specific. In some embodiments, the metabolite may be associated with hepatic encephalopathy, e.g. , bilirubin. Non- limiting examples of molecules or metabolites include, e.g. , bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese in their blood and intestines. Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein. In some embodiments, the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g. , RNS or ROS promoter. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut or tumor microenvironment, e.g., arabinose and tetracycline.

[0107] "Exogenous environmental condition(s)" refer to setting(s) or circumstance(s) under which the promoter described herein is induced. The phrase "exogenous environmental conditions" is meant to refer to the environmental conditions external to the engineered micororganism, but endogenous or native to the host subject environment. Thus, "exogenous" and "endogenous" may be used interchangeably to refer to environmental conditions in which the environmental conditions are

endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous

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

[0108] An "oxygen level-dependent promoter" or "oxygen level-dependent regulatory region" refers to a nucleic acid sequence to which one or more oxygen level- sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.

[0109] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et ah , 2009; Eiglmeier et ah , 1989; Galimand et ah, 1991 ; Hasegawa et al, 1998; Hoeren et al, 1993; Salmon et al, 2003), and non-limiting examples are shown in Table 1.

[0110] In a non-limiting example, a promoter (PfnrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz,

2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression.

However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS.

Table 1. Examples of transcription factors and responsive genes and regulatory regions

[0111] As used herein, a "gene cassette" or "operon" encoding a biosynthetic pathway refers to the two or more genes that are required to produce a desired metabolite or protein(s) of interest. Non-limiting examples of such metabolites include, e.g., tryptophan and/or kynurenine, or produce a gut barrier function enhancer molecule, e.g., butyrate, propionate. Proteins of interest may for example be a checkpoint inhibitor. In addition to encoding a set of genes capable of producing said molecule, the gene cassette or operon may also comprise additional transcription and translation elements, e.g., a ribosome binding site. [0112] As used herein, the term "low oxygen" is meant to refer to a level, amount, or concentration of oxygen (0₂) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., <21% O_2; <160 torr 0₂₎). Thus, the term "low oxygen condition or conditions" or "low oxygen environment" refers to conditions or environments containing lower levels of oxygen than are present in the atmosphere. In some embodiments, the term "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0₂) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term "low oxygen" is meant to refer to a level, amount, or concentration of 0₂ that is 0-60 mmHg 0₂ (0-60 torr 0₂₎ (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg 0₂), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg 0₂, 0.75 mmHg 0₂, 1.25 mmHg 0₂, 2.175 mmHg 0₂, 3.45 mmHg 0₂, 3.75 mmHg 0₂, 4.5 mmHg 0₂, 6.8 mmHg 0₂, 11.35 mmHg 02, 46.3 mmHg 0₂, 58.75 mmHg, etc., which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way). In some embodiments, "low oxygen" refers to about 60 mmHg 0₂ or less (e.g., 0 to about 60 mmHg 0₂₎. The term "low oxygen" may also refer to a range of 0₂ levels, amounts, or concentrations between 0-60 mmHg 0₂ (inclusive), e.g., 0-5 mmHg 0₂, < 1.5 mmHg 0₂, 6-10 mmHg, < 8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way. See, for example, Albenberg et al., Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al., J Clin. Invest., 41(11): 1971- 1980 (1962); Crompton et al., J Exp. Biol., 43: 473-478 (1965); He et al., PNAS (USA), 96: 4586-4591 (1999);

McKeown, Br. J. Radiol., 87:20130676 (2014) (doi: 10.1259/brj.20130676), each of which discusses the oxygen levels found in the mammalian gut of various species and each of which are incorportated by reference herewith in their entireties. In some embodiments, the term "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0₂) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments, "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0₂) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions. For example, Table A summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (0₂) is expressed as the amount of dissolved oxygen ("DO") which refers to the level of free, non-compound oxygen (0₂) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; lmg/L = 1 ppm), or in micromoles (umole) (1 umole 0₂ = 0.022391 mg/L 0₂). Fondriest Environmental, Inc., "Dissolved Oxygen", Fundamentals of Environmental Measurements, 19 Nov 2013,

www. fondriest. com/environmental- measurements/parameters/water-quality/dissolved- oxygen/>. In some embodiments, the term "low oxygen" is meant to refer to a level, amount, or concentration of oxygen (0₂) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way. The level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (0₂) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium). Well-aerated solutions (e.g., solutions subjected to mixing and/or stirring) without oxygen producers or consumers are 100% air saturated. In some embodiments, the term "low oxygen" is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-40%, inclusive (e.g., 0-5%, 0.05 - 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. In some embodiments, the term "low oxygen" is meant to refer to 9% 0₂ saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, 0₂ saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of 0₂ saturation levels between 0-9%, inclusive (e.g., 0-5%, 0.05 - 0.1%, 0.1-0.2%, 0.1- 0.5%, 0.5 - 2.0%, 0-8%, 5-7%, 0.3-4.2% 0_2, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.

Table 2. Intestinal Oxygen Tension

[0113] As used herein, "kynureninase" refers to an enzyme that enzyme that catalyses the cleavage of kynurenine (KYN) into anthranilic acid (AA). It can also act on 3-hydroxykynurenine (3HK) (to produce 3-hydroxyanthranillic acid, 3HAA) and some other (3-arylcarbonyl)-alanines. In some embodiments, "kynureninase" refers to the human form of the enzyme. In other embodiments, the kynureninase is of bacterial origin, e.g. Pseudomonas luminescens. Human and bacterial enzymes differ in their preferred substrates. In the human pathway along one arm, kynurenine is hydroxylated by a flavoenzymes monooxygenase to give 30HK, which is the preferred substrate for human kynureninase. In contrast, the bacterial kynureninase acts preferentially on KYN itself, leading to the generation of anthranilate. The human enzyme also performs this reaction, although KYN is a less preferred substrate as compared ot 3HK (Phillips, Structure and mechanism of kynureninase.. Arch Biochem Biophys. 2014 Feb

15;544:69-74). A triple mutation in the active site of the human enzyme

(H102W/S332G/N333T), as described in Philips 2014, gave rise to an enzyme which had measurable activity with KYN, but no measurable activity with 3HT. In some embodiments, of the disclosure the genetically engineered bacteria may express this triple mutant human enzyme.

[0114] "kyurenine aminotransferase" or "KAT" refers to an enzyme which transaminates and cyclizes KYN to KYNA. In some embodiments, the genetically engineered bacteria comprise KAT, and can modulate metabolite ratios, including but not limited to the KYNA:QUIN ratios. Four KATs have been reported in mammalian brains, KAT I/glutamine transaminase K/cysteine conjugate beta- lyase 1, KAT

II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase.

[0115] As used herein, a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et ah , 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, "non-native" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non- native nucleic acid sequence, e.g. , gene or gene cassette, may be present on a plasmid or bacterial chromosome. In some embodiments, the genetically engineered bacteria of the invention comprise a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g. , a FNR- responsive promoter operably linked to a butyrogenic gene cassette, or an arginine production cassette. In addition, multiple copies of the gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same non-native nucleic acid sequence, e.g. , gene, gene cassette, or regulatory region, in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.

[0116] "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive

Escherichia coli oS promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli σ32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ70 promoter (e.g. , lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter

(BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105),

M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter

(BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis σΑ promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG

(BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis σΒ promoter (e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella

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

BBa_Kl 13010; BBa_Kl 13011 ; BBa_Kl 13012; BBa_R0085; BBa_R0180;

BBa_R0181 ; BBa_R0182; BBa_R0183 ; BBa_Z0251 ; BBa_Z0252; BBa_Z0253)), a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof.

[0117] "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e. , the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.

[0118] As used herein, the term "gene sequence" is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. The gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also menat to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence.

[0119] "Tumor-targeting bacteria" refer to bacteria that are capable of directing themselves to cancerous cells. Tumor-targeting bacteria may be naturally capable of directing themselves to cancerous cells, necrotic tissues, and/or hypoxic tissues. In some embodiments, bacteria that are not naturally capable of directing themselves to cancerous cells, necrotic tissues, and/or hypoxic tissues are genetically engineered to direct themselves to cancerous cells, necrotic tissues, and/or hypoxic tissues. Tumor- targeting bacteria may be further engineered to enhance or improve desired biological properties, mitigate systemic toxicity, and/or ensure clinical safety. These species, strains, and/or subtypes may be attenuated, e.g., deleted for a toxin gene. In some embodiments, tumor-targeting bacteria have low infection capabilities. In some embodiments, tumor-targeting bacteria are motile. In some embodiments, the tumor- targeting bacteria are capable of penetrating deeply into the tumor, where standard treatments do not reach. In some embodiments, tumor-targeting bacteria are capable of colonizing at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a malignant tumor. Examples of tumor-targeting bacteria include, but are not limited to, Bifidobacterium, Caulobacter, Clostridium, Escherichia coli, Listeria, Mycobacterium, Salmonella, Streptococcus, and Vibrio, e.g., Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55, Clostridium butyricum miyairi, Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-ΝΎ, Clostridium paraputrificum, Clostridium pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, and Vibrio cholera (Cronin et al, 2012; Forbes, 2006; Jain and Forbes, 2001; Liu et al., 2014; Morrissey et al., 2010; Nuno et al., 2013; Patyar et al., 2010; Cronin, et al., Mol Ther 2010; 18: 1397-407). In some embodiments, the tumor-targeting bacteria are non-pathogenic bacteria.

[0120] "Tumor-targeting oncolytic virus" refer to virus that are capable of directing themselves to cancerous cells. Tumor-targeting virus may be naturally capable of directing themselves to cancerous cells, necrotic tissues, and/or hypoxic tissues. Oncolytic viruses that are not naturally capable of directing themselves to cancerous cells, necrotic tissues, and/or hypoxic tissues can be genetically engineered to direct themselves to cancerous cells, necrotic tissues, and/or hypoxic tissues. In addition, they can be further engineered to target specific cancer or cell types. Tumor- targeting oncolytic viruses may also be engineered to enhance or improve desired biological properties (e.g., lytic properties), mitigate systemic toxicity, and/or ensure clinical safety. These species, strains, and/or subtypes may be attenuated, e.g., deleted for a toxin gene. In some embodiments, tumor-targeting bacteria have low infection capabilities. Examples of tumor-targeting oncolytic viruses are provided elsewhere herein and are reviewed in Chlocca et al., Cancer Immunol research, 2014, 2:295-300 and Kaufman, et al., Nature, 2016, 14:642-662.

[0121] "Microorganism" refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell.

Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, protozoa, and yeast. In some aspects, the microorganism is engineered ("engineered microorganism") to produce one or more payloads or therapeutic molecules. In certain aspects, the microorganism is engineered to import and/or catabolize certain toxic metabolites, substrates, or other compounds from its environment, e.g., the gut or the tumor micorenvironment. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites, molecules, or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus. [0122] As used herein, the term "recombinant microorganism" refers to a microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state. Thus, a "recombinant bacterial cell" or "recombinant bacteria" refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

[0123] A "programmed or engineered microorganism" refers to a

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

[0124] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum,

Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et ah, 2009; Dinleyici et ah, 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589, 168; U.S. Patent No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.

[0125] As used herein, "payload" refers to one or more polynucleotides and/or polypeptides of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus. In some embodiments, the payload is encoded by a gene or multiple genes or an operon. In some embodiments, the one or more genes and/or operon(s) comprising the payload are endogenous to the microorganism. In some embodiments, the one or more elements of the payload is derived from a different microorganism and/or organism. In some embodiments, the payload is a therapeutic payload. In some embodiments, the payload is encoded by genes for the biosynthesis of a molecule. In some embodiments, the payload is encoded by genes for the metabolism, catabolism, or degradation of a molecule. In some embodiments, the payload is encoded by genes for the importation of a molecule. In some embodiments, the payload is encoded by genes for the exportation of a molecule. In some embodiments, the payload is a regulatory molecule(s), e.g. , a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload expression is driven from an inducible promoter, such as from FNRS. In some embodiments, payload expression is driven from a consitutitve promoter. In some embodiments, the payload comprises a repressor element, such as a kill switch. In alternate embodiments, the payload is produced by a bio synthetic or biochemical pathway, wherein the bio synthetic or biochemical pathway may optionally be endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads.

[0126] An "anti-cancer molecule" refers to one or more therapeutic substances or drugs of interest to be produced by a genetically engineered microorganism, e.g. , engineered bacteria or engineered oncolytic virus, which are capable of reducing and/or inhibiting cell growth or replication. In some embodiments, the anti-cancer molecule is a therapeutic molecule that is useful for modulating or treating a cancer. In some embodiments, the anti-cancer molecule is a therapeutic molecule encoded by a gene. In alternate embodiments, the anti-cancer molecule is a therapeutic molecule produced by a biochemical or bio synthetic pathway, wherein the bio synthetic or biochemical pathway may optionally be endogenous to the microorganism. In some embodiments, the genetically engineered microorganism is capable of producing two or more anticancer molecules. Non-limiting examples of anti-cancer molecules include immune checkpoint inhibitors (e.g., CTLA-4 antibodies, PD- 1 antibodies, PDL- 1 antibodies), cytotoxic agents (e.g., Cly A, FASL, TRAIL, TNF-alpha), immuno stimulatory cytokines and co-stimulatory molecules (e.g., OX40, CD28, ICOS, CCL21, IL-2, IL- 18, IL- 15, IL- 12, IFN-gamma, IL-21, TNFs, GM-CSF), antigens and antibodies (e.g. , tumor antigens, neoantigens, CtxB-PSA fusion protein, CPV-OmpA fusion protein, NY-ESO- 1 tumor antigen, RAF1, antibodies against immune suppressor molecules, anti-VEGF, Anti-CXR4/CXCL12, anti-GLPl, anti-GLP2, anti-galectinl, anti-galectin3, anti-Tie2, anti-CD47, antibodies against immune checkpoints, antibodies against

immunosuppressive cytokines and chemokines), DNA transfer vectors (e.g., endostatin, thrombospondin- 1, TRAIL, SMAC, Stat3, Bcl2, FLT3L, GM-CSF, IL- 12, AFP, VEGFR2), and enzymes (e.g. , E. coli CD, HSV-TK). In some embodiments, the anticancer molecule includes nucleic acid molecules that mediate RNA interference, microRNA response or inhibition, TLR response, antisense gene regulation, target protein binding (aptamer or decoy oligos), gene editing, such as CRISPR interference. In some embodiments, bacteria or virus can be used as vectors to transfer DNA into mammalian cells, e.g. , by bactofection (Bernardes et ah, 2013). In some embodiments, the genetically engineered bacteria comprising gene sequences comprising one or more circuits for the production or catabolism of tryptophan and/or one of its metabolites further comprise gene sequences for the expression of one or more anti-cancer molecules.

[0127] As used herein, "anti-inflammation molecules" and/or "gut barrier function enhancer molecules" include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP- 1, IL- 10, IL-27, TGF-βΙ, TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD₂, and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein. Such molecules may also include compounds that inhibit pro-inflammatory molecules, e.g., a single- chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-γ, IL- Ιβ, IL-6, IL-8, IL- 17, and/or chemokines, e.g., CXCL-8 and CCL2. Such molecules also include AHR agonists (e.g., which result in IL-22 production, e.g., indole acetic acid, indole-3-aldehyde, and indole) and and PXR agonists (e.g., IP A), as described herein. Such molecules also include HDAC inhibitors (e.g., butyrate), activators of GPR41 and/or GPR43 (e.g., butyrate and/or propionate and/or acetate), activtators of GPR109A (e.g., butyrate), inhibitors of NF-kappaB signaling (e.g., butyrate), and modulators of PPARgamma (e.g., butyrate), activators of AMPK signaling (e.g., acetate), and modulators of GLP-1 secretion. Such molecules also include hydroxyl radical scavengers and antioxidants (e.g., IP A). A molecule may be primarily anti- inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2. A molecule may be both anti- inflammatory and gut barrier function enhancing. An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene. Alternatively, an anti- inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., butyrate. In some embodiments, the genetically engineered bacteria comprising gene sequences comprising one or more circuits for the production or catabolism of tryptophan and/or one of its metabolites further comprise gene sequences for the expression of one or more anti-inflammation molecules and/or gut barrier function enhancer molecules.

[0128] "Metabolic effector molecules" and/or "satiety effector molecules" include, but are not limited to, n-acyl-phophatidylethanolamines (NAPEs), n-acyl- ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP- 2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). Such molecules may also include compounds that inhibit a molecule that promotes metabolic disease, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor. A metabolic and/or satiety effector molecule may be encoded by a single gene, e.g., glucogon-like peptide 1 is encoded by the GLP-1 gene. In some embodiments, the genetically engineered bacteria comprising gene sequences comprising one or more circuits for the production or catabolism of tryptophan and/or one of its metabolites further comprise gene sequences for the expression of one or more metabolic effector molecule and/or satiety effector molecules.

[0129] "Probiotic" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an

appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et ah, 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al, 2012; Cuevas-Ramos et ah, 2010; Olier et ah, 2012; Nougayrede et al., 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

[0130] As used herein, "stably maintained" or "stable" bacterium is used to refer to a bacterial host cell carrying non- native genetic material, e.g., a feedback resistant argA gene, mutant arginine repressor, and/or other mutant arginine regulon that is incorporated into the host genome or propagated on a self-rep Heating extra- chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium may be a genetically engineered bacterium comprising an argA^ gene, in which the plasmid or chromosome carrying the argA^ gene is stably maintained in the bacterium, such that argA^ can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo.

[0131] As used herein, the term "modulate" and its cognates means to alter, regulate, or adjust positively or negatively a molecular or physiological readout, outcome, or process, to effect a change in said readout, outcome, or process as compared to a normal, average, wild-type, or baseline measurement. Thus, for example, "modulate" or "modulation" includes up-regulation and down-regulation. A non- limiting example of modulating a readout, outcome, or process is effecting a change or alteration in the normal or baseline functioning, activity, expression, or secretion of a biomolecule (e.g. a protein, enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid, or other compound). Another non- limiting example of modulating a readout, outcome, or process is effecting a change in the amount or level of a biomolecule of interest, e.g. in the serum and/or the gut lumen. In another non- limiting example, modulating a readout, outcome, or process relates to a phenotypic change or alteration in one or more disease symptoms. Thus, "modulate" is used to refer to an increase, decrease, masking, altering, overriding or restoring the normal functioning, activity, or levels of a readout, outcome or process (e.g, biomolecule of interest, and/or molecular or physiological process, and/or a phenotypic change in one or more disease symptoms).

[0132] As used herein, the term "treat" and its cognates refer to an amelioration of a disease, disorder, and/or condition described herein, or at least one discernible symptom thereof. In another embodiment, "treat" refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "treat" refers to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g. , stabilization of a discernible symptom), physiologically (e.g. , stabilization of a physical parameter), or both. In another embodiment, "treat" refers to slowing the progression or reversing the progression of a disease, disorder, and/or condition. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition. Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder.

[0133] As used herein a "pharmaceutical composition" refers to a preparation of genetically engineered bacteria of the invention with other components such as a physiologically suitable carrier and/or excipient. [0134] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.

[0135] The term "excipient" refers to an inert substance added to a

pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[0136] The terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g.,

hyperammonemia. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder associated with elevated ammonia concentrations. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

[0137] As used herein, the term "conventional treatment" or "conventional cancer therapy" refers to treatment or therapy that is widely accepted and used by most healthcare professionals. It is different from alternative or complementary therapies, which are not as widely used. Examples of conventional treatment, e.g. , for cancer, include surgery, chemotherapy, targeted therapies, radiation therapy, tomotherapy, immunotherapy, cancer vaccines, hormone therapy, hyperthermia, stem cell transplant (peripheral blood, bone marrow, and cord blood transplants), photodynamic therapy, therapy, and blood product donation and transfusion.

[0138] An antibody generally refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen. An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond. The recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The N- terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively.

[0139] As used herein, the term "antibody" or "antibodies"is meant to encompasses all variations of antibody and fragments thereof that possess one or more particular binding specificities. Thus, the term "antibody" or "antibodies" is meant to include full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single domain antibodies (sdAB, VHH framents), heavy chain antibodies (HCAb), nanobodies, diabodies, and minibodies. Antibodies can have more than one binding specificity, e.g. be bispecific. The term "antibody" is also meant to include so- called antibody mimetics. Antibody mimetics refers to small molecules, e.g., 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure. Antibody mimetics, include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B

crystalline), Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from Sulfolobus acidocaldarius), Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif), Fynomers (SH3 domain of Fyn), Kunitz domain peptides Kunitz domains of various protease inhibitors), Ecallantide (Kalbitor), and Monobodies. In certain aspects, the term

"antibody" or "antibodies" is meant to refer to a single chain antibody(ies), single domain antibody(ies), and camelid antibody(ies). Utility of antibodies in the treatment of cancer and additional anti cancer antibodies can for example be found in Scott et ah, Antibody Therapy for Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference in its entirety.

[0140] A "single-chain antibody" or "single-chain antibodies" typically refers to a peptide comprising a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and optionally a linker or bond, such as a disulfide bond. The single- chain antibody lacks the constant Fc region found in traditional antibodies. In some embodiments, the single-chain antibody is a naturally occurring single-chain antibody, e.g., a camelid antibody. In some embodiments, the single-chain antibody is a synthetic, engineered, or modified single-chain antibody. In some embodiments, the single-chain antibody is capable of retaining substantially the same antigen specificity as compared to the original immunoglobulin despite the addition of a linker and the removal of the constant regions. In some aspects, the single chain antibody can be a "scFv antibody", which refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins (without any constant regions), optionally connected with a short linker peptide of ten to about 25 amino acids, as described, for example, in U.S. Patent No. 4,946,778, the contents of which is herein incorporated by reference in its entirety. The Fv fragment is the smallest fragment that holds a binding site of an antibody, which binding site may, in some aspects, maintain the specificity of the original antibody. Techniques for the production of single chain antibodies are described in U.S. Patent No. 4,946,778. The Vh and VL sequences of the scFv can be connected via the N-terminus of the VH connecting to the C-terminus of the VL or via the C-terminus of the VH connecting to the N-terminus of the VL. ScFv fragments are independent folding entities that can be fused indistinctively on either end to other epitope tags or protein domains. Linkers of varying length can be used to link the Vh and VL sequences, which the linkers can be glycine rich (provides flexibility) and serine or threonine rich (increases solubility). Short linkers may prevent association of the two domains and can result in multimers (diabodies, tribodies, etc.). Long linkers may result in proteolysis or weak domain association (described in Voelkel et al el., 2011). Linkers of length between 15 and 20 amino acids or 18 and 20 amino acids are most often used. Additional non- limiting examples of linkers, including other flexible linkers are described in Chen et al., 2013 (Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Fusion Protein Linkers: Property, Design and Functionality), the contents of which is herein incorporated by reference in its entirety. Flexible linkers are also rich in small or polar amino acids such as Glycine and Serine, but can contain additional amino acids such as Threonine and Alanine to maintain flexibility, as well as polar amino acids such as Lysine and Glutamate to improve solubility. Exemplary linkers include, but are not limited to, (Gly-Gly-Gly-Gly-Ser)n, KESGSVSSEQLAQFRSLD and

EGKSSGSGSESKST, (Gly)8, and Gly and Ser rich flexible linker, GSAGSAAGSGEF. "Single chain antibodies" as used herein also include single-domain antibodies, which include camelid antibodies and other heavy chain antibodies, light chain antibodies, including nanobodies and single domains VH or VL domains derived from human, mouse or other species. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies include domain antigen-binding units which have a camelid scaffold, derived from camels, llamas, or alpacas. Camelids produce functional antibodies devoid of light chains. The heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen- binding molecules (Fabs) or single chain variable fragments (scFvs). Camelid antibodies are capable of attaining binding affinities comparable to those of

conventional antibodies. Camelid scaffold-based antibodies can be produced using methods well known in the art. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. Alternatively, the dimeric variable domains from IgG from humans or mice can be split into monomers. Nanobodies are single chain antibodies derived from light chains. The term "single chain antibody" also refers to antibody mimetics.

[0141] In some embodiments, the antibodies expressed by the engineered microorganisms are bispecfic. In certain embodiments, a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope. Antigen- binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Monomeric single-chain diabodies (scDb) are readily assembled in bacterial and mammalian cells and show improved stability under physiological conditions (Voelkel et ah, 2001 and references therein; Protein Eng. (2001) 14 (10): 815-823 (describes optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies).

[0142] As used herein, the term "polypeptide" includes "polypeptide" as well as "polypeptides," and refers to a molecule composed of amino acid monomers linearly linked by amide bonds {i.e., peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, "peptides," "dipeptides," "tripeptides, "oligopeptides," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term

"polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation,

phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term "peptide" or "polypeptide" may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.

[0143] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms "fragment," "variant," "derivative" and "analog" include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the

corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non- naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0144] Polypeptides also include fusion proteins. As used herein, the term "variant" includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term "fusion protein" refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins. "Derivatives" include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution.

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

Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, He, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu. [0145] As used herein, the term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones.

[0146] As used herein the term "linker", "linker peptide" or "peptide linkers" or "linker" refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g. , that link two polypeptide domains. As used herein the term "synthetic" refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.

[0147] As used herein the term "codon-optimized sequence" refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism.

[0148] Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA

(mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0149] As used herein, the terms "secretion system" or "secretion protein" refers to a native or non-native secretion mechanism capable of secreting or exporting the protein of interest or therapeutic protein from the microbial, e.g. , bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g. HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g. , hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the protein(s) of interest or therapeutic protein(s) include a "secretion tag" of either RNA or peptide origin to direct the protein(s) of interest or therapeutic protein(s) to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the protein(s) of interest or therapeutic protein(s) from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g. , OmpT cleavage thereby releasing the protein(s) of interest or therapeutic protein(s) into the extracellular milieu.

[0150] As used herein, the term "transporter" is meant to refer to a mechanism, e.g. , protein or proteins, for importing a molecule, e.g. , amino acid, toxin, metabolite, substrate, etc. into the microorganism from the extracellular milieu.

[0151] An "immune checkpoint inhibitor" or "immune checkpoint" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more immune checkpoint proteins. Immune checkpoint proteins regulate T-cell activation or function, and are known in the art. Non-limiting examples include CTLA- 4 and its ligands CD 80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2.

Immune checkpoint proteins are responsible for co- stimulatory or inhibitory interactions of T-cell responses, and regulate and maintain self-tolerance and physiological immune responses. Systemic immunotherapy, e.g., using CTLA-4 inhibitors, may alter immunoregulation, provoke immune dysfunction, and result in opportunistic

autoimmune disorders (see, e.g., Kong et al, 2014).

[0152] As used herein, a genetically engineered microorganism, e.g., engineered bacterium or engineered oncolytiv virus, or anti-cancer molecule that "inhibits" cancerous cells refers to a bacterium or virus or molecule that is capable of reducing cell proliferation, reducing tumor growth, and/or reducing tumor volume by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to control, e.g., an untreated control or an unmodified microorganism of the same subtype under the same conditions.

[0153] As used herein, a genetically engineered microorganism, e.g., engineered bacterium or engineered oncolytic virus, or molecule that "inhibits" a biological molecule refers to a bacterium or virus or molecule that is capable of reducing, decreasing, or eliminating the biological activity, biological function, and/or number of that biological moleculer, as compared to control, e.g., an untreated control or an unmodified microorganism of the same subtype under the same conditions.

[0154] As used herein, a genetically engineered microorganism, e.g., engineered bacterium or engineered oncolytic virus, or molecule that "activates" or "stimulates" a biological molecule, refers to a bacterium or virus or anti-cancer molecule that is capable of activating, increasing, enhancing, or promoting the biological activity, biological function, and/or number of that biological molecule, as compared to control, e.g., an untreated control or an unmodified microorganism of the same subtype under the same conditions.

[0155] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0156] The phrase "and/or," when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and/or C" indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase "and/or" may be used interchangeably with "at least one of or "one or more of the elements in a list.

[0157] Bacteria

[0158] The genetically engineered bacteria disclosed herein are capable of modulating tryptophan metabolism. In some embodiments, the genetically engineered bacteria are naturally non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis,

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

[0159] In some embodiments, the genetically engineered bacteria are

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

[0160] In some embodiments, the genetically engineered bacteria are capable of targeting cancerous cells, particularly in the hypoxic regions of a tumor. Im some embodiments, the bacteria can produce an anti-cancer molecule, e.g., kynurenine, alone or in combination with other anti-cancer molecules provided herein In some

embodiments, the genetically engineered bacterium is a tumor-targeting bacterium that expresses an anti-cancer molecule or a gene cassette under the control of a promoter that is activated by low-oxygen conditions, e.g., the hypoxic environment of a tumor.

[0161] In some embodiments, the tumor-targeting microorganism is a bacterium that is naturally capable of directing itself to cancerous cells, necrotic tissues, and/or hypoxic tissues. For example, bacterial colonization of tumors may be achieved without any specific genetic modifications in the bacteria or in the host (Yu et al., 2008). In some embodiments, the tumor-targeting bacterium is a bacterium that is not naturally capable of directing itself to cancerous cells, necrotic tissues, and/or hypoxic tissues, but is genetically engineered to do so. In some embodiments, the genetically engineered bacteria spread hematogenously to reach the targeted tumor(s). Bacterial infection has been linked to tumor regression (Hall, 1998; Nauts and McLaren, 1990), and certain bacterial species have been shown to localize to and lyse necrotic mammalian tumors (Jain and Forbes, 2001). Non- limiting examples of tumor-targeting bacteria are shown in Table 3.

Table 3. Bacteria with tumor-targeting capability

Caulobacter spp cancer." Oncology letters 8.6 (2014): 2359- Clostridium spp 2366.

Escherichia coli MG1655 Cronin, Michelle, et al. "High resolution in Escherichia coli Nissle vivo bio luminescent imaging for the study Bifidobacterium breve UCC2003 of bacterial tumour targeting." PloS one 7.1 Salmonella typhimurium (2012): e30940.; Zhou, et al, Med

Hypotheses. 2011 Apr;76(4):533-4. doi: 10.1016/j.mehy.2010.12.010. Epub 2011 Jan 21; Zhang et al., Appl Environ

Microbiol. 2012 Nov; 78(21): 7603-7610; Danino et al. ,

ScienceTranslationalMedicine, 2015 Vol 7 Issue 289, pp. 289ra84

Clostridium novyi-NT Bernardes, Nuno, Ananda M. Chakrabarty, Bifidobacterium spp and Arsenio M. Fialho. "Engineering of Mycobacterium bovis bacterial strains and their products for Listeria monocytogenes cancer therapy." Applied microbiology and Escherichia coli biotechnology 97.12 (2013): 5189-5199. Salmonella spp

Salmonella typhimurium

Salmonella choleraesuis Patyar, S., et al. "Bacteria in cancer Vibrio cholera therapy: a novel experimental strategy." Listeria monocytogenes Biomed Sci 17.1 (2010): 21-30.

Escherichia coli

Bifidobacterium adolescentis

Clostridium acetobutylicum

Salmonella typhimurium

Clostridium histolyticum

Escherichia coli Nissle 1917 Danino et al. "Programmable probiotics for detection of cancer in urine." Sci Transl Med. 2015 May 27;7(289):289ra84

[0162] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be modified and adapted for other species, strains, and subtypes of bacteria.

[0163] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum

(Sonnenborn et al., 2009). The residence time of bacteria in vivo can be determined using the methods described herein. In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention. [0164] The tumor-targeting capability of certain bacteria appears to be dependent on the stage of tumor development, but independent of tumor type (Yu et al., 2008). Intravenously injected bacteria have been shown to target the central portion of tumors and coincide with the necrotic regions of those tumors (Yu et al., 2008).

Inflammation alone has been shown to be insufficient to sustain bacterial colonization (Yu et al, 2008). In some embodiments, tumors are sensitized, e.g., by oncolytic vaccinia virus, prior to bacterial delivery to enhance colonization. In some

embodiments, the blood-borne bacteria enter tumors and are able to amplify in the central necrotic region because clearance of bacteria is inhibited (Yu et al., 2008).

[0165] In some embodiments, the gene of interest or gene cassette is expressed in a bacterium which enhances the efficacy of immunotherapy. Vetizou et al (2015) describe T cell responses specific for Bacteroides thetaiotaomicron or Bacteroides fragilis that were associated with the efficacy of CTLA-4 blockade in mice and in patients. Sivan et al. (2015) illustrate the importance of Bifidobacterium to antitumor immunity and anti-PD-Ll antibody against (PD-1 ligand) efficacy in a mouse model of melanoma. In some embodiments, the bacteria expressing one or more genes or gene cassettes are Bacteroides. In some embodiments, the bacteria expressing the one or more anticancer molecules are Bifidobacterium. In some embodiments, the bacteria expressing one or more genes or gene cassettes are Escherichia Coli Nissle. In some embodiments, the bacteria expressing one or more genes or gene cassettes are

Clostridium novyi-NT. In some embodiments, the bacteria expressing one or more genes or gene cassettes are Clostridium butyricum miyairi.

[0166] In certain embodiments, the genetically engineered bacteria are obligate anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are facultative anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are aerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive bacteria and lack LPS. In some embodiments, the genetically engineered bacteria are Gram-negative bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and obligate anaerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and facultative anaerobic bacteria. In some embodiments, the genetically engineered bacteria are nonpathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria, Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis,

Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron,

Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve

UCC2003, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55, Clostridium butyricum miyairi, Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-ΝΎ,

Clostridium paraputrificum, Clostridium pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, Vibrio cholera, and the bacteria shown in Table 2. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis,

Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis. In some embodiments, Lactobacillus is used for tumor- specific delivery of one or more anti-cancer molecules. Lactobacillus casei injected intravenously has been found to accumulate in tumors, which was enhanced through nitroglycerin (NG), a commonly used NO donor, likely due to the role of NO in increasing the blood flow to hypo vascular tumors (Fang et al, 2016 (Methods Mol Biol. 2016;1409:9-23. Enhancement of Tumor-Targeted Delivery of Bacteria with Nitroglycerin Involving Augmentation of the EPR Effect). [0167] In some embodiments, the genetically engineered bacteria are obligate anaerobes. In some embodiments, the genetically engineered bacteria are Clostridia and capable of tumor- specific delivery of anti-cancer molecules. Clostridia are obligate anaerobic bacterium that produce spores and are naturally capable of colonizing and in some cases lysing hypoxic tumors (Groot et al, 2007). In experimental models, Clostridia have been used to deliver pro-drug converting enzymes and enhance radiotherapy (Groot et al., 2007). In some embodiments, the genetically engineered bacteria is selected from the group consisting of Clostridium novyi-NT, Clostridium histolyticium, Clostridium tetani, Clostridium oncolyticum, Clostridium sporogenes, and Clostridium beijerinckii (Liu et al., 2014). In some embodiments, the Clostridium is naturally non-pathogenic. For example, Clostridium oncolyticum is apathogenic and capable of lysing tumor cells. In alternate embodiments, the Clostridium is naturally pathogenic but modified to reduce or eliminate pathogenicity. For example,

Clostridium novyi are naturally pathogenic, and Clostridium novyi-NT are modified to remove lethal toxins. Clostridium novyi-NT and Clostridium sporogenes have been used to deliver single-chain HIF-Ια antibodies to treat cancer and is an "excellent tumor colonizing Clostridium strains" (Groot et al., 2007).

[0168] In some embodiments, the genetically engineered bacteria facultative anaerobes. In some embodiments, the genetically engineered bacteria are Salmonella, e.g., Salmonella typhimurium, and are capable of tumor- specific delivery of anti-cancer molecules. Salmonella are no n- spore-forming Gram-negative bacteria that are facultative anaerobes. In some embodiments, the Salmonella are naturally pathogenic but modified to reduce or eliminate pathogenicity. For example, Salmonella

typhimurium is modified to remove pathogenic sites (attenuated). In some

embodiments, the genetically engineered bacteria are Bifidobacterium and capable of tumor- specific delivery of anti-cancer molecules. Bifidobacterium are Gram-positive, branched anaerobic bacteria. In some embodiments, the Bifidobacterium is naturally non-pathogenic. In alternate embodiments, the Bifidobacterium is naturally pathogenic but modified to reduce or eliminate pathogenicity. Bifidobacterium and Salmonella have been shown to preferentially target and replicate in the hypoxic and necrotic regions of tumors (Yu et al., 2014).

[0169] In some embodiments, the genetically engineered bacteria are Gram- negative bacteria. In some embodiments, the genetically engineered bacteria are E. coli. For example, E. coli Nissle has been shown to preferentially colonize tumor tissue in vivo following either oral or intravenous administration (Zhang et al., 2012 and Danino et al., 2015). E. coli have also been shown to exhibit robust tumor- specific replication (Yu et al, 2008). In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the Enterobacteriaceae family that "has evolved into one of the best characterized probiotics" (Ukena et al., 2007). The strain is characterized by its complete

harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added).

[0170] The genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in tissues or blood serum (Sonnenborn et al., 2009). In some embodiments, the genetically engineered bacteria are administered repeatedly. In some embodiments, the genetically engineered bacteria are administered once.

[0171] In certain embodiments, the anti-cancer molecule (s) described herein are expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the anti-cancer molecule is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria. One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be modified and adapted for other species, strains, and subtypes of bacteria.

[0172] Further examples of bacteria which are suitable are described in

International Patent Publication WO/2014/043593, the contents of which is herein incorporated by reference in its entirety. In some embodiments, such bacteria are mutated to attenuate one or more virulence factors.

[0173] In some aspects, the engineered bacteria can be combined with other therapies, e.g., conventional therapies, and immunotherapies or anti- inflammatory therapies, as the case may be.

Regulating expression of tryptophan metabolism enzymes molecules

[0174] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut or the tumor microenvironment. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.

[0175] Payload (and/or polypeptides of interest and/or proteins of interest and/or therapeutic polypeptides and/or therapeutic proteins and/or therapeutic peptides and/or effector and/or effector molecules) include any of the metabolites described herein and/or any of the enzyme(s) or polypeptide(s) which function as enzymes for the production or catabolism of such effector molecules. Effector molecules and payloads include but are not limited to anti-cancer molecules, immune modulators, gut barrier enhancer molecules, ant i- inflammatory molecules, satiety molecules or

neuromodulatory effectors. Non-limiting examples of payloads include kynureninase, tryptophan production enzymes, tryptophan degradation enzymes, one or more kynurenine production enzymes, serotonin or melatonin production or degradation enzymes, indole metabolite production or degradation enzymes (described herein) KP metabolite production or degradation enzymes and others described herein. As used herein, the term "gene of interest" or "gene sequence of interest" includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more effector molecules and payloads include but are not limited to anti-cancer molecules, immune modulators, gut barrier enhancer molecules, ant i- inflammatory molecules, satiety molecules or effectors, neuromodulatory molecules described herein, e.g., kynureninase, tryptophan production enzymes, tryptophan degradation enzymes, one or more kynurenine production enzymes, serotonin or melatonin production or degradation enzymes, indole metabolite production or degradation enzymes (described herein) KP metabolite production or degradation enzymes and others described herein.

[0176] In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.

[0177] In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.

[0178] In some embodiments, the genetically engineered bacteria comprise two or more payloads, all of which are present on the chromosome. In some embodiments, the genetically engineered bacteria comprise two or more payloads, all of which are present on one or more same or different plasmids. In some embodiments, the genetically engineered bacteria comprise two or more payloads, some of which are present on the chromosome and some of which are present on one or more same or different plasmids.

[0179] In any of the nucleic acid embodiments, described above, the one or more payload(s) for producing the a polypeptide of interest combinations are operably linked to one or more directly or indirectly inducible promoter(s). In some

embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under exogeneous environmental conditions, e.g., conditions found in the gut, the tumor microenvironment, or other tissue specific conditions. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced by metabolites found in the gut, the tumor microenvironment, or other specific conditions. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under inflammatory conditions (e.g., RNS, ROS), as described herein. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under immunosuppressive conditions, e.g., as found in the tumor, or other specific tissues, as described herein. In some

embodiments, the two or more gene sequence(s) are linked to a directly or indirectly inducible promoter that is induced by exposure a chemical or nutritional inducer, which may or may not be present under in vivo conditions and which may be present during in vitro conditions (such as strain culture, expansion, manufacture), such as tetracycline or arabinose, or others described herein. In some embodiments, the two or more payloads are all linked to a constitutive promoter. Such constitutive promoters are described in Table 48 - Table 58 herein.

[0180] In a non- limiting example, the genetically engineered bacteria may comprise two payloads, one of which is linked to a constitutive promoter, and one of which is linked to a directly or indirectly inducible promoter. In a non-limiting example, the genetically engineered bacteria may comprise three payloads, one of which is linked to a constitutive promoter, and one of which is linked to a directly or indirectly inducible promoter and one of which is linked to a second, different directly or indirectly inducible promoter.

[0181] In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is induced under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.

[0182] In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions). In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions).

[0183] In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous

environmental conditions specific to the hypoxic environment of a tumor and/or the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the hypoxic environment of a tumor and/or the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the tumor, a particular tissue, or the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is coadministered with the bacterial cell.

FNR dependent Regulation

[0184] The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a polypeptide of interest, wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the inducible promoter is an oxygen level-dependent promoter and a polypeptide of interest is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, in low oxygen conditions, the oxygen level-dependent promoter is activated by a corresponding oxygen level- sensing transcription factor, thereby driving production of the polypeptide of interest.

[0185] Bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An oxygen level-dependent promoter is a nucleic acid sequence to which one or more oxygen level- sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. In one embodiment, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter. In a more specific aspect, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, such as the hypoxic

environment of a tumor and/or the environment of the mammalian gut, and/or other specific tissues.

[0186] In certain embodiments, the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In E. coli, FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et ah, 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in Table 4A and Table 4B below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.

Table 4A. FNR Promoter Sequences

Table 4B. FNR Promoter sequences

GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATC

nirBl

AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC SEQ ID NO: 8

AATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCA ATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAG AAATCGAGGCAAAA

CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTAC

AGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCCTGTTA

GGTTTCGTCAGCCGTCACCGTCAGCATAACACCCTGACCTCTCATT

AATTGCTCATGCCGGACGGCACTATCGTCGTCCGGCCTTTTCCTCT

nirBl

CTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTC SEQ ID NO: 9

TATTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAG

AAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATATAAA

GGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAG

GTAGGCGGTAATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaa ggagatatacat

GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATC

nirB3

SEQ ID NO: 10 AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC

AATATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCA ATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAG AAATCGAGGCAAAA

ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTAT

ydfz GGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAA

SEQ ID NO: 11 AAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTG

GGCTTCGACTGTAAATCAGAAAGGAGAAAACACCT

GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATC

nirB+RBS

AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC SEQ ID NO: 12

AATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCA ATAAGCGGGGTTGCTGAATCGTTAAGGATCCCTCTAGAAATAATT TTGTTTAACTTTAAGAAGGAGATATACAT

CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTA

ydfZ+RBS TGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACA SEQ ID NO: 13 AAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCC

CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT

GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC

fnrSl

CGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC SEQ ID NO: 14

AATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTAACTTTAA

GAAGGAGATATACAT

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT

GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC

fnrS2

CGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC SEQ ID NO: 15

AATATCTCTCTTGGATCCAAAGTGAACTCTAGAAATAATTTTGTTT

AACTTTAAGAAGGAGATATACAT

TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGT CAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCA CTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTA

nirB+crp

TTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAA SEQ ID NO: 16 TATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAAT

ATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATA AGCGGGGTTGCTGAATCGTTAAGGTAGaaatgtgatctagttcacatttGCGGTA ATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaaggagatatacat

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT

fnrS+crp GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC SEQ ID NO: 17 CGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC

AATATCTCTCaaatgtgatctagttcacatttiiigiitoflciitoflgflflggflgfltotocfli

[0187] FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.

[0188] Non-limiting FNR promoter sequences are provided in Table 4 and

Table 5. Table 4 and Table 5 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence.

Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, nirB l promoter (SEQ ID NO: 8), nirB2 promoter (SEQ ID NO: 9), nirB3 promoter (SEQ ID NO: 10), ydfZ promoter (SEQ ID NO: 11), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 12), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 13), fnrS, an anaerobically induced small RNA gene (fnrS l promoter SEQ ID NO: 14 or fnrS2 promoter SEQ ID NO: 15), nirB promoter fused to a crp binding site (SEQ ID NO: 16), and fnrS fused to a crp binding site (SEQ ID NO: 17). In some embodiments, the FNR-responsive promoter is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of any one of SEQ ID NOs: 1-17.

[0189] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al, 2010) or ANR (Ray et al., 1997). In these embodiments, expression of the payload gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.

[0190] In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the payload expressed under the control of anaerobic regulation of arginine deiminiase and nitrate reduction transcriptional regulator (ANR). In P. aeruginosa, ANR is "required for the expression of

physiological functions which are inducible under oxygen-limiting or anaerobic conditions" (Winteler et al., 1996; Sawers 1991). P. aeruginosa ANR is homologous with E. coli FNR, and "the consensus FNR site (TTGAT— ATCAA) was recognized efficiently by ANR and FNR" (Winteler et al, 1996). Like FNR, in the anaerobic state, ANR activates numerous genes responsible for adapting to anaerobic growth. In the aerobic state, ANR is inactive. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all have functional analogs of ANR (Zimmermann et al., 1991). Promoters that are regulated by ANR are known in the art, e.g., the promoter of the arcDABC operon {see, e.g., Hasegawa et al., 1998).

[0191] In other embodiments, the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Gorke and Stiilke, 2008). In some embodiments, the gene or gene cassette for producing a payload molecule is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter (e.g. , an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing an payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g. , by adding glucose to growth media in vitro.

[0192] In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level- sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level- sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen- level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g. , one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic

environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g. , Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity. [0193] In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen- level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic

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

[0194] In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the gene encoding the payload are present on different plasmids. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid.

[0195] In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the gene encoding the payload are present on the same chromosome. In some instances, it may be

advantageous to express the oxygen level- sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region.

RNS -dependent regulation

[0196] In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.

[0197] As used herein, "reactive nitrogen species" and "RNS" are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO*), peroxynitrite or peroxynitrite anion (ONOO-), nitrogen dioxide (·Ν02), dinitrogen trioxide (N203), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOC02-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

[0198] As used herein, "RNS-inducible regulatory region" refers to a nucleic acid sequence to which one or more RNS -sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS- inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s), e.g. , any of the payloads described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

[0199] As used herein, "RNS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more RNS -sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS- derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g. , a payload gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

[0200] As used herein, "RNS-repressible regulatory region" refers to a nucleic acid sequence to which one or more RNS -sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the

transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

[0201] As used herein, a "RNS-responsive regulatory region" refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS- derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 7.

Table 5. Examples of RNS-sensing transcription factors and RNS-responsive genes

[0202] In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS- sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated.

[0203] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0204] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR "is an NO-responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide" (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload(s).

[0205] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR

(dissimilatory nitrate respiration regulator) "promotes the expression of the nir, the nor and the nos genes" in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads. In some embodiments, the DNR is Pseudomonas aeruginosa DNR. [0206] In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing a payload molecule expressed under the control of the dissimilatory nitrate respiration regulator (DNR). DNR is a member of the FNR family (Arai et al., 1995) and is a transcriptional regulator that is required in conjunction with ANR for "anaerobic nitrate respiration of Pseudomonas aeruginosa" (Hasegawa et al., 1998). For certain genes, the FNR-binding motifs "are probably recognized only by DNR" (Hasegawa et al., 1998). Any suitable transcriptional regulator that is controlled by exogenous environmental conditions and corresponding regulatory region may be used. Non- limiting examples include ArcA/B, ResD/E, NreA/B/C, and AirSR, and others are known in the art.

[0207] In some embodiments, the tunable regulatory region is a RNS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0208] In some embodiments, the tunable regulatory region is a RNS- derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is "an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism" (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s).

[0209] In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some

embodiments, the genetically engineered bacterium of the invention expresses a RNS- sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the

Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[0210] In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0211] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., encoding a payload. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments, include, but are not limited to, TetR, CI, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a payload gene or genes is expressed.

[0212] A RNS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS- responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012).

[0213] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS- sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of therapeutic molecule. In some embodiments, expression of the RNS- sensing transcription factor is controlled by the same promoter that controls expression of therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0214] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0215] In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity.

[0216] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on different plasmids. In some

embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS- sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on the same chromosome.

[0217] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g., the NsrR gene, and a corresponding regulatory region, e.g., a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS -responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS.

[0218] In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0219] In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more encoding a payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

[0220] In some embodiments, the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50- fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800- fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g., by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen).

[0221] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900- fold, at least about 1, 000-fold, or at least about 1, 500-fold more of payload in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments, using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.

RQS -dependent regulation

[0222] In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the payload is expressed under the control of an cellular damaged- dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.

[0223] As used herein, "reactive oxygen species" and "ROS" are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal- catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H202), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical (·ΟΗ), superoxide or superoxide anion (·02-), singlet oxygen (102), ozone (03), carbonate radical, peroxide or peroxyl radical (·02-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOCl), nitric oxide (NO*), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al, 2014). [0224] As used herein, "ROS-inducible regulatory region" refers to a nucleic acid sequence to which one or more ROS -sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS- inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g. , a sequence or sequences encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS- inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

[0225] As used herein, "ROS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more ROS -sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS- derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g. , one or more genes encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

[0226] As used herein, "ROS-repressible regulatory region" refers to a nucleic acid sequence to which one or more ROS -sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the

transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

[0227] As used herein, a "ROS-responsive regulatory region" refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS- derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 6.

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

[0228] In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated.

[0229] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0230] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR

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

[0231] In alternate embodiments, the tunable regulatory region is a ROS- inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is "activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression" (Koo et al., 2003). "SoxR is known to respond primarily to superoxide and nitric oxide" (Koo et al, 2003), and is also capable of responding to H202. The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al, 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing a payload.

[0232] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0233] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR "binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event," but oxidized OhrR is "unable to bind its DNA target" (Duarte et al, 2010). OhrR is a "transcriptional repressor [that] ... senses both organic peroxides and NaOCl" (Dubbs et al, 2012) and is "weakly activated by H202 but it shows much higher reactivity for organic hydroperoxides" (Duarte et al, 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a payload gene. In the presence of ROS, e.g., NaOCl, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing a payload.

[0234] OhrR is a member of the MarR family of ROS -responsive regulators. "Most members of the MarR family are transcriptional repressors and often bind to the - 10 or -35 region in the promoter causing a steric inhibition of RNA polymerase binding" (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some

embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012).

[0235] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is "a MarR-type transcriptional regulator" that binds to an "18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA" and is "reversibly inhibited by the oxidant H202" (Bussmann et al., 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to "a putative polyisoprenoid-binding protein (eg 1322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cgl426), two putative FMN reductases (cgl l50 and cgl850), and four putative monooxygenases (cg0823, cgl848, cg2329, and cg3084)" (Bussmann et al, 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al, 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g. , a payload. In the presence of ROS, e.g. , H202, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload.

[0236] In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some

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

[0237] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0238] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR "when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis

(hemAXCDBL, fur, and zoaA) and its own synthesis (perR)" (Marinho et ah, 2014). PerR is a "global regulator that responds primarily to H202" (Dubbs et ah , 2012) and "interacts with DNA at the per box, a specific palindromic consensus sequence

(TTATAATNATTATAA) residing within and near the promoter sequences of PerR- controlled genes" (Marinho et ah, 2014). PerR is capable of binding a regulatory region that "overlaps part of the promoter or is immediately downstream from it" (Dubbs et al, 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et ah, 2012).

[0239] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these

embodiments, include, but are not limited to, TetR, CI, and LexA. In some

embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed.

[0240] A ROS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although "OxyR is primarily thought of as a transcriptional activator under oxidizing conditions . . . OxyR can function as either a repressor or activator under both oxidizing and reducing conditions" (Dubbs et ah, 2012), and OxyR "has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)" (Zheng et ah, 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et ah, 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by RosR. In addition, "PerR- mediated positive regulation has also been observed...and appears to involve PerR binding to distant upstream sites" (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by PerR.

[0241] One or more types of ROS-sensing transcription factors and

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

[0242] Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 7. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or a functional fragment thereof.

Table 7. Nucleotide sequences of exemplary OxyR-regulated regulatory regions

Regulatory

Sequence

sequence Regulatory

Sequence

sequence

TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACA

GAGCACAAAATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGT

TATCAGCCTTGTTTTCTCCCTCATTACTTGAAGGATATGAAGCTA

katG AAACCCTTTTTTATAAAGCATTTGTCCGAATTCGGACATAATCA (SEQ ID AAAAAGCTTAATTAAGATCAATTTGATCTACATCTCTTTAACCA NO: 18) ACAATATGTAAGATCTCAACTATCGCATCCGTGGATTAATTCAA

TTATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACTGTA

GAGGGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAAGGT

ACC

TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACG

dps

CTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCGGA

(SEQ ID

ACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGACA NO: 19)

TAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGAGA AAGGTACC

GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC

CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG

CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA

ahpC

CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC

(SEQ ID

AAAGTTCACAAAGTTGTCTTACGAAGGTTGTAAGGTAAAACTTA NO: 20)

TCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAAATTG

GTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATGCG

AATTCATTAAAGAGGAGAAAGGTACC

CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGCG

oxyS

ATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTCTG

(SEQ ID

ACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGTA NO: 21)

CC

[0243] In some embodiments, the regulatory region sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and/or SEQ ID NO: 21.

[0244] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS- sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of therapeutic molecule. In some embodiments, expression of the ROS- sensing transcription factor is controlled by the same promoter that controls expression of therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0245] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0246] In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate

embodiments, the native ROS-sensing transcription factor, e.g. , OxyR, is deleted or mutated to reduce or eliminate wild-type activity.

[0247] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on different plasmids. In some

embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing therapeutic molecule are present on the same chromosome.

[0248] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g. , the soxR gene, and a corresponding regulatory region, e.g. , a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS -responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of ROS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS.

[0249] In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0250] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s). In some

embodiments, the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS -responsive regulatory region. In some embodiments, the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS-responsive regulatory region.

[0251] Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a

corresponding transcription factor.

[0252] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the payload. In some embodiments, the gene encoding the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the payload is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the payload. In some embodiments, the gene encoding the payload is expressed on a chromosome.

Propionate and other promoters

[0253] In some embodiments, the genetically engineered bacteria comprise the gene or gene cassette for producing payload expressed under the control of an inducible promoter that is responsive to specific molecules or metabolites in the environment, e.g., the tumor microenvironment, a specific tissue, or the mammalian gut. For example, the short-chain fatty acid propionate is a major microbial fermentation metabolite localized to the gut (Hosseini et al., 2011). In one embodiment, the gene or gene cassette for producing a payload is under the control of a propionate-inducible promoter. In a more specific embodiment, the gene or gene cassette for producing the payload is under the control of a propionate-inducible promoter that is activated by the presence of propionate in the mammalian gut. Any molecule or metabolite found in the mammalian gut, in a healthy and/or disease state, may be used to induce payload expression. Non-limiting examples of inducers include propionate, bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese. In alternate embodiments, the gene or gene cassette for producing therapeutic polypeptide is under the control of a pBAD promoter, which is activated in the presence of the sugar arabinose.

[0254] In some embodiments, the gene or gene cassette for producing the polypeptide of interest is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing polypeptide of interest is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing a polypeptide of interest is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the to the tumor and/or the mammalian gut. In some embodiments, the gene or gene cassette for producing polypeptide of interest is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the tumor and/or the mammalian gut. In some embodiments, the gene or gene cassette for producing polypeptide of interest is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing polypeptide of interest is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0255] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene or gene cassette for producing the polypeptide of interest, such that the gene or gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut or the tumor microenvironment. In some embodiments, a bacterium may comprise multiple copies of the gene or gene cassette for producing a polypeptide of interest. In some embodiments, gene or gene cassette for producing the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, gene or gene cassette for producing a polypeptide of interest is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing gene or gene cassette expression. In some embodiments, gene or gene cassette for producing a polypeptide of interest is expressed on a chromosome.

[0256] Table 42 lists a propionate promoter sequence. In some embodiments, the propionate promoter is induced in the mammalian gut. In some embodiments, the propionate promoter sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of SEQ ID NO: 22.

Table 8. Propionate promoter sequence

AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCATTGAGGA AGTCAGCTTTCCCGCTTTTGCCAGAGCCTGTAATACATCGAA TCCGCTGGGTTTGATGAGGATGACAGGTACCGACAGTCGGCT TTTTAAATAAGCGCCGTTGGAACCTGCCGCGATAATCGCGTC GCAGCGTTCGGTTGCCAGTTTTTTGCGAATGTAGGCTACTGC CTTTTCAAAACCGAGCTGAATAGGCGTGATCGTCGCCAGATG ATCAAACTCCAGGCTGATATCCCGAAATAGTTCGAACAGGCG CGTTACCGAGACCGTCCAGATCACCGGTTTATCGCTATTATC GCGCGAAGCGCTATGCACAGTAACCATCGTCGTAGAT TCATG TTTAAGGAACGAATTCTTGTTTTATAGATGTTTCGTTAATGT TGCAATGAAACACAGGCCTCCGTTTCATGAAACGTTAGCTGA CTCGTTTTTCTTGTGACTCGTCTGTCAGTATTAAAAAAGATT TTTCATTTAACTGATTGTTTTTAAATTGAATTTTATTTAATG GTTTCTCGGTTTTTGGGTCTGGCATATCCCTTGCTTTAATGA GTGCATCTTAATTAACAATTCAATAACAAGAGGGCTGAATag taatttcaacaaaataacgagcattcgaatg

Other Inducible Promoters

[0257] In some embodiments, the gene encoding the a polypeptide of interest is present on a plasmid and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the gene encoding the a polypeptide of interest is present in the chromosome and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).

[0258] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the one or more gene sequences(s), inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s), encoding the a polypeptide of interest, such that the a polypeptide of interest can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the tumor or in the gut. In some embodiments, bacterial cell comprises two or more distinct copies of the one or more gene sequences(s) encoding a polypeptide of interest, which is controlled by a promoter inducible one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same one or more gene sequences(s) encoding a polypeptide of interest, which is controlled by a promoter inducible one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the one or more gene sequences(s) encoding a polypeptide of interest(s), is present on a plasmid and operably linked to a directly or indirectly inducible promoter inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the one or more gene sequences(s) encoding a polypeptide of interest, is present on a chromosome and operably linked to a directly or indirectly inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).

[0259] In some embodiments, one or more gene sequence(s) encoding polypeptides of interest described herein is present on a plasmid and operably linked to promoter a directly or indirectly inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene encoding a polypeptide of interest, which is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s), such that a polypeptide of interest can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., under culture conditions, and/or in vivo, e.g., in the gut or the tumor microenvironment. In some embodiments, bacterial cell comprises two or more gene sequence(s) for the production of a polypeptide of interest, one or more of which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same gene sequence(s) for the production of a polypeptide of interest which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of different gene sequence(s) for the production of a polypeptide of interest, one or more of which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).

[0260] In some embodiments, the gene sequence(s) for the production of a polypeptide of interest is present on a plasmid and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, gene sequence(s) for the production of a polypeptide of interest is present in the chromosome and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).

[0261] In some embodiments, the promoter that is operably linked to the gene encoding the polypeptide of interest is directly or indirectly induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). [0262] In some embodiments, one or more inducible promoter(s) are useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, the promoters are induced during in vivo expression of one or more anticancer, satiety, gut barrier enhancer, immune modulatory and/or neuromodulatory molecules and/or other polypeptide(s) of interest. In some embodiments, expression of one or more a polypeptide of interest(s) and/or other polypeptide(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a chemical and/or nutritional inducer and/or metabolite which is co- administered with the genetically engineered bacteria of the invention.

[0263] In some embodiments, expression of one or more a polypeptide of interest and/or other polypeptide(s) of interest, is driven directly or indirectly by one or more promoter(s) induced by a chemical and/or nutritional inducer and/or metabolite during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the promoter(s) induced by a chemical and/or nutritional inducer and/or metabolite are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with a polypeptide of interest(s) and/or other polypeptide(s) of interest prior to administration. In some embodiments, the cultures, which are induced by a chemical and/or nutritional inducer and/or metabolite, are grown aerobically. In some embodiments, the cultures, which are induced by a chemical and/or nutritional inducer and/or metabolite, are grown anaerobically.

[0264] The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub- saturating L-arabinose concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L- arabinose transporter (Zhang et al., Development and Application of an Arabinose- Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum; Appl. Environ. Microbiol. August 2012 vol. 78 no. 16 5831-5838).

Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

[0265] In one embodiment, expression of one or more polypeptides of interest, e.g., one or more therapeutic polypeptide(s), is driven directly or indirectly by one or more arabinose inducible promoter(s).

[0266] In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some

embodiments, the promoter is directly or indirectly induced by a molecule that is coadministered with the genetically engineered bacteria of the invention, e.g., arabinose.

[0267] In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., arabinose. In some

embodiments, the cultures, which are induced by arabinose, are grown arerobically. In some embodiments, the cultures, which are induced by arabinose, are grown

anaerobically.

[0268] In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some

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

[0269] In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0270] In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter

[0271] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 23. In some embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 24. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 25. [0272] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter.

[0273] In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some

embodiments, the promoter is directly or indirectly induced by a molecule that is coadministered with the genetically engineered bacteria of the invention, e.g., rhamnose

[0274] In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., rhamnose. In some

embodiments, the cultures, which are induced by rhamnose, are grown arerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobically.

[0275] In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some

embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, conditions of the tumor microenvironment, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s).

[0276] In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a construct which is integrated into the bacterial chromosome.

Exemplary insertion sites are described herein.

[0277] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 26. [0278] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-l- thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydro lyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacl) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by E. coli, its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate Lacl, can be used instead of IPTG in a similar manner.

[0279] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s).

[0280] In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., IPTG.

[0281] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown arerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically.

[0282] In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non- limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, conditions of the tumor microenvironment, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene

sequence(s).

[0283] In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0284] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 27. In some embodiments, the IPTG inducible construct further comprises a gene encoding lacl, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 28. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 29.

[0285] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracyclme-responsi ve promoters. Gossen M & Bujard ll.PNAS, 1992 Jun 15:89(12):5547-51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. Tetracycline- controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16) from herpes simplex virus. In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline- controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.

[0286] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s). In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co -administered with the genetically engineered bacteria of the invention, e.g., tetracycline

[0287] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically.

[0288] In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some

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

[0289] In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. [0290] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the bolded sequences of SEQ ID NO: 34 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 34 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 34 in italics (Tet repressor is in italics).

[0291] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et al., Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy; J Biotechnol. 2015 Jun 10; 203: 32-40, and references therein). Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage λ promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the λ promoters can then be efficiently regulated by the mutant thermo labile cI857 repressor of bacteriophage λ. At temperatures below 37 °C, cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in FIG. 88A. Inducible expression from the ParaBad can be controlled or further fine- tuned through the optimization of the ribosome binding site (RBS), as described herein.

[0292] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). In some embodiments, thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., temperature.

[0293] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shup off production of the one or more protein(s) of interest. This can be done in a thermoregulated system by growing the strain at lower temperatures, e.g., 30 C. Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically.

[0294] In one embodiment, thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein

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

[0295] In some embodiments, thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0296] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 30. In some embodiments, thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 31. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 33.

[0297] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.

[0298] This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. FIG. 89A depicts a schematic of the gene organization of a PssB promoter.

[0299] In one embodiment, expression of one or more protein(s) of interest is indirectly regulated by a repressor expressed under the control of one or more PssB promoter(s).

[0300] In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest. In some

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

[0301] In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0302] In another non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The

chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic conditions, e.g., the gut and/or conditions of the tumor microenvironment, but prevent survival under aerobic conditions (biosafety switch). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 35.

[0303] Sequences useful for expression from inducible promoters are listed in Table 9. Table 9. Inducible promoter construct sequences

26

Lac Promoter ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATG region CCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCG

CTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGA

SEQ ID NO: TCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATG 27 TGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATG

TGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCT

CTACAAATAATTTTGTTTAAAACAACACCCACTAAGATAA

CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA

CAT

LacO GGAATTGTGAGCGCTCACAATT

Lacl (in reverse TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC orientation) TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT

GCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA

SEQ ID NO: GACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA 28 GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA

GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA

ACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAG

ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC

GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT

CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT

TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT

CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATG

CCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAA

TGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG

ACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGG

AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATC

AAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCAC

AGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATC

AGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG

CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC

ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCG

CCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG

AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAG

TTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC

ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG

GCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG

ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT

TCAT

Lacl MKP VTLYD V AE Y AG VS YQT VS RV VNQ AS H VS AKTRE KVE A polypeptide AM AELNYIPNRV AQQLAGKQS LLIGV ATS SLALH APS QIVAA sequence IKS R ADQLG AS V V VS M VERS G VE AC KA A VHNLL AQRVS GLI

INYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT

SEQ ID NO: RLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRN 29 QIQPIAEREGDWS AMS GFQQTMQMLNEGIVPT AMLV ANDQ

MALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIK QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLA PNTQTASPRALADSLMQLARQVSRLESGQ

Region ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC comprising GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTG Temperature CATAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCC sensitive GTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAA promoter CAACACCCACTAAGATAACTCTAGAAATAATTTTGTTTAA

CTTTAAGAAGGAGATATACAT

SEQ ID NO:

30

mutant cI857 TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACT repressor TTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTG

GGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCT

SEQ ID NO: ATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCA 31 AGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCT

CAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGG

CTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACC

TCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGC

TTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGC

TTAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACA

GGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAA

CCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGG

GCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGTAAGCA

ATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAA

TAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCT

GCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTT

TTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGC

TCTTGTGTTAATGGTTTCTTTTTTGTGCTCAT

RBS and leader CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA region CAT

SEQ ID NO:

32

mutant cI857 MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKM repressor GMGQS G VG ALFNGIN ALN A YN A ALLTKILK VS VEEFS PS I AR polypeptide EI YEM YE A VS MQPS LRS E YE YP VFS H VQ AGMFS PKLRTFT KG sequence D AERWVS TTKKAS DS AFWLE VEGNS MT APT GS KPS FPDGML

ILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNP

SEQ ID NO: QYPMIPCNESCSVVGKVIASQWPEETFG

33

TetR-Tet Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataa promoter gaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcata construct ctatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacct SEQ ID NO: aaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttgg 34 cataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgta cttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaat cttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggct aaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacaccta gcttctgggcgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcg cigitoaicaciitociiitoictoaictogacaicattaattcctaatttttgttgacactctatcattg atagagttattttaccactccctatcagtgatagagaaaagtgaacicto^aaatoaiiii^iii aactttaagaaggagatatacat PssB promoter tcacctttcccggattaaacgcttttttgcccggtggcatggtgctaccggcgatcacaaacggtta attatgacacaaattgacctgaatgaatatacagtattggaatgcattacccggagtgttgtgtaac

SEQ ID NO: aatgtctggccaggtttgtttcccggaaccgaggtcacaacatagtaaaagcgctattggtaatgg 35 tacaatcgcgcgtttacacttattc

Constitutive promoters

[0304] In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.

[0305] In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut and/or conditions of the tumor microenvironment, as described herein. In some embodiments, the promoters is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut and/or conditions of the tumor microenvironment, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.

[0306] In some embodiments, the constitutive promoter that is operably linked to the gene encoding the payload is active in various exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions).

[0307] In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the gut of a mammal and/or specific to conditions of the tumor microenvironment. In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoter is active in low-oxygen or anaerobic conditions such as the environment of the mammalian gut and/or conditions of the tumor microenvironment. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites that are specific to the gut of a mammal and/or conditions of the tumor microenvironment. In some embodiments, the constitutive promoter is directly or indirectly induced by a molecule that is coadministered with the bacterial cell. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.

[0308] Bacterial constitutive promoters are known in the art. Examplary constitutive promoters are listed in the following Tables.

[0309]

Table 10. Constitutive E. coli σ70 promoters

BBa_J23105 constitutive 35 SEO ID NO: 609 promoter family ggctagctcagtcctaggt

member actatgctagc

BBa_J23106 constitutive 35 SEO ID NO: 610 promoter family ggctagctcagtcctaggt

member atagtgctagc

BBa_J23107 constitutive 35 SEO ID NO: 611 promoter family ggctagctcagccctaggt

member attatgctagc

BBa_J23108 constitutive 35 SEO ID NO: 612 promoter family agctagctcagtcctaggt

member ataatgctagc

BBa_J23109 constitutive 35 SEO ID NO: 613 promoter family agctagctcagtcctaggg

member actgtgctagc

BBa_J23110 constitutive 35 SEO ID NO: 614 promoter family ggctagctcagtcctaggt

member acaatgctagc

BBa_J23111 constitutive 35 SEO ID NO: 615 promoter family ggctagctcagtcctaggt

member atagtgctagc

BBa_J23112 constitutive 35 SEO ID NO: 616 promoter family agctagctcagtcctaggg

member attatgctagc

BBa_J23113 constitutive 35 SEO ID NO: 617 promoter family ggctagctcagtcctaggg

member attatgctagc

BBa_J23114 constitutive 35 SEO ID NO: 618 promoter family ggctagctcagtcctaggt

member acaatgctagc

BBa_J23115 constitutive 35 SEO ID NO: 619 promoter family agctagctcagcccttggt

member acaatgctagc

BBa_J23116 constitutive 35 SEO ID NO: 620 promoter family agctagctcagtcctaggg

member actatgctagc

BBa_J23117 constitutive 35 SEO ID NO: 621 promoter family agctagctcagtcctaggg

member attgtgctagc

BBa_J23118 constitutive 35 SEO ID NO: 622 promoter family ggctagctcagtcctaggt

member attgtgctagc

BBa_J23119 constitutive 35 SEO ID NO: 623 promoter family agctagctcagtcctaggt member ataatgctagc

BBa_J23150 lbp mutant from 35 SEO ID NO: 624 J23107 ggctagctcagtcctaggt

attatgctagc

BBa_J23151 lbp mutant from 35 SEO ID NO: 625 J23114 ggctagctcagtcctaggt

acaatgctagc

BBa_J44002 pBAD reverse 130 SEO ID NO: 626 aaagtgtgacgccgtgcaa

ataatcaatgt

BBa_J48104 NikR promoter, a 40 SEO ID NO: 627 protein of the ribbon gacgaatacttaaaatcgtc

helix-helix family of atacttattt trancription factors

that repress expre

BBa_J54200 lacq_Promoter 50 SEO ID NO: 628 aaacctttcgcggtatggc

atgatagcgcc

BBa_J56015 lacIQ - promoter 57 SEO ID NO: 629 sequence tgatagcgcccggaagag

agtcaattcagg

BBa_J64951 E. coli CreABCD 81 SEO ID NO: 630 phosphate sensing ttatttaccgtgacgaacta

operon promoter attgctcgtg

BBa_K088007 GlnRS promoter 38 SEO ID NO: 631 catacgccgttatacgttgtt

tacgctttg

BBa_Kl 19000 Constitutive weak 38 SEO ID NO: 632 promoter of lacZ ttatgcttccggctcgtatgt

tgtgtggac

BBa_Kl 19001 Mutated LacZ 38 SEO ID NO: 633 promoter ttatgcttccggctcgtatg

gtgtgtggac

BBa_Kl 330002 Constitutive 35 SEO ID NO: 634 promoter (J23105) ggctagctcagtcctaggt

actatgctagc

BBa_Kl 37029 constitutive 39 SEO ID NO: 635 promoter with atatatatatatatataatgg

(TA)10 between -10 aagcgtttt and -35 elements

BBa_Kl 37030 constitutive 37 SEO ID NO: 636 promoter with atatatatatatatataatgg

(TA)9 between -10 aagcgtttt and -35 elements BBa_Kl 37031 constitutive 62 SEO ID NO: 637 promoter with (C)10 ccccgaaagcttaagaata

between -10 and -35 taattgtaagc elements

BBa_Kl 37032 constitutive 64 SEO ID NO: 638 promoter with (C)12 ccccgaaagcttaagaata

between -10 and -35 taattgtaagc elements

BBa_K137085 optimized (TA) 31 SEO ID NO: 639 repeat constitutive tgacaatatatatatatatat

promoter with 13 bp aatgctagc between -10 and -35

elements

BBa_K137086 optimized (TA) 33 SEO ID NO: 640 repeat constitutive acaatatatatatatatatata

promoter with 15 bp atgctagc between -10 and -35

elements

BBa_K137087 optimized (TA) 35 SEO ID NO: 641 repeat constitutive aatatatatatatatatatata

promoter with 17 bp atgctagc between -10 and -35

elements

BBa_K137088 optimized (TA) 37 SEO ID NO: 642 repeat constitutive tatatatatatatatatatata

promoter with 19 bp atgctagc between -10 and -35

elements

BBa_K137089 optimized (TA) 39 SEO ID NO: 643 repeat constitutive tatatatatatatatatatata

promoter with 21 bp atgctagc between -10 and -35

elements

BBa_Kl 37090 optimized (A) 35 SEO ID NO: 644 repeat constitutive aaaaaaaaaaaaaaaaaat

promoter with 17 bp ataatgctagc between -10 and -35

elements

BBa_Kl 37091 optimized (A) 36 SEO ID NO: 645 repeat constitutive aaaaaaaaaaaaaaaaaat

promoter with 18 bp ataatgctagc between -10 and -35

elements

BBa_K1585100 Anderson Promoter 78 SEO ID NO: 646 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585101 Anderson Promoter 78 SEO ID NO: 647 with lacl binding ggaattgtgagcggataac site aatttcacaca

BBa_K1585102 Anderson Promoter 78 SEO ID NO: 648 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585103 Anderson Promoter 78 SEO ID NO: 649 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585104 Anderson Promoter 78 SEO ID NO: 650 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585105 Anderson Promoter 78 SEO ID NO: 651 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585106 Anderson Promoter 78 SEO ID NO: 652 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585110 Anderson Promoter 78 SEO ID NO: 653 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585113 Anderson Promoter 78 SEO ID NO: 654 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585115 Anderson Promoter 78 SEO ID NO: 655 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585116 Anderson Promoter 78 SEO ID NO: 656 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585117 Anderson Promoter 78 SEO ID NO: 657 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585118 Anderson Promoter 78 SEO ID NO: 658 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_K1585119 Anderson Promoter 78 SEO ID NO: 659 with lacl binding ggaattgtgagcggataac

site aatttcacaca

BBa_Kl 824896 J23100 + RBS 88 SEO ID NO: 660 gattaaagaggagaaatac

tagagtactag

BBa_K256002 J23101:GFP 918 SEO ID NO: 661 caccttcgggtgggcctttc

tgcgtttata BBa_K256018 J23119:IFP 1167 SEO ID NO: 662 caccttcgggtgggcctttc

tgcgtttata

BBa_K256020 J23119:H01 949 SEO ID NO: 663 caccttcgggtgggcctttc

tgcgtttata

BBa_K256033 Infrared signal 2124 SEO ID NO: 664 reporter caccttcgggtgggcctttc

(J23119:IFP:J23119 tgcgtttata :H01)

BBa_K292000 Double terminator + 138 SEO ID NO: 665 constitutive ggctagctcagtcctaggt

promoter acagtgctagc

BBa_K292001 Double terminator + 161 SEO ID NO: 666 Constitutive tgctagctactagagattaa

promoter + Strong agaggagaaa RBS

BBa_K418000 IPTG inducible Lac 1416 SEO ID NO: 667 promoter cassette ttgtgagcggataacaaga

tactgagcaca

BBa_K418002 IPTG inducible Lac 1414 SEO ID NO: 668 promoter cassette ttgtgagcggataacaaga

tactgagcaca

BBa_K418003 IPTG inducible Lac 1416 SEO ID NO: 669 promoter cassette ttgtgagcggataacaaga

tactgagcaca

BBa_K823004 Anderson promoter 35 SEO ID NO: 670 J23100 ggctagctcagtcctaggt

acagtgctagc

BBa_K823005 Anderson promoter 35 SEO ID NO: 671 J23101 agctagctcagtcctaggt

attatgctagc

BBa_K823006 Anderson promoter 35 SEO ID NO: 672 J23102 agctagctcagtcctaggt

actgtgctagc

BBa_K823007 Anderson promoter 35 SEO ID NO: 673 J23103 agctagctcagtcctaggg

attatgctagc

BBa_K823008 Anderson promoter 35 SEO ID NO: 674 J23106 ggctagctcagtcctaggt

atagtgctagc

BBa_K823010 Anderson promoter 35 SEO ID NO: 675 J23113 ggctagctcagtcctaggg

attatgctagc BBa_K823011 Anderson promoter 35 SEO ID NO: 676 J23114 ggctagctcagtcctaggt

acaatgctagc

BBa_K823013 Anderson promoter 35 SEO ID NO: 677 J23117 agctagctcagtcctaggg

attgtgctagc

BBa_K823014 Anderson promoter 35 SEO ID NO: 678 J23118 ggctagctcagtcctaggt

attgtgctagc

BBa_M13101 M13K07 gene I 47 SEO ID NO: 679 promoter cctgtttttatgttattctctct

gtaaagg

BBa_M13102 M13K07 gene II 48 SEO ID NO: 680 promoter aaatatttgcttatacaatctt

cctgtttt

BBa_M13103 M13K07 gene III 48 SEO ID NO: 681 promoter gctgataaaccgatacaatt

aaaggctcct

BBa_M13104 M13K07 gene IV 49 SEO ID NO: 682 promoter ctcttctcagcgtcttaatct

aagctatcg

BBa_M13105 M13K07 gene V 50 SEO ID NO: 683 promoter atgagccagttcttaaaatc

gcataaggta

BBa_M13106 M13K07 gene VI 49 SEO ID NO: 684 promoter ctattgattgtgacaaaata

aacttattcc

BBa_M13108 M13K07 gene VIII 47 SEO ID NO: 685 promoter gtttcgcgcttggtataatc

gctgggggtc

BBa_M13110 M13110 48 SEO ID NO: 686 ctttgcttctgactataatagt

cagggtaa

BBa_M31519 Modified promoter 60 SEO ID NO: 687 sequence of g3. aaaccgatacaattaaagg

ctcctgctagc

BBa_R1074 Constitutive 74 SEO ID NO: 688 Promoter I caccacactgatagtgcta

gtgtagatcac

BBa_R1075 Constitutive 49 SEO ID NO: 689 Promoter II gccggaataactccctata

atgcgccacca

BBa_S03331 —Specify Parts List- ttgacaagcttttcctcagct

SEO ID NO: 690 ccgtaaact Table 11. Constitutive E. coli σ promoters

BBa K143010

Promoter etc for B. subtilis . . . atccttatcgttatgggtattgtttgtaat 56 SEO ID NO: 702

BBa K143011 Promoter gsiB for B.

38 SEO ID NO: 703 subtilis taaaagaattgtgagcgggaatacaacaac

BBa K143013 Promoter 43 a constitutive

56 SEO ID NO: 704 promoter for B. subtilis aaaaaaagcgcgcgattatgtaaaatataa

Table 15. Constitutive promoters from miscellaneous prokaryotes

Name Description Promoter Sequence Length

BBa Kl 12706

Pspv2 from Salmonella . . . tacaaaataattcccctgcaaacattatca 474 SEO ID NO: 705

BBa Kl 12707

Pspv from Salmonella . . . tacaaaataattcccctgcaaacattatcg 1956 SEO ID NO: 706

Table 16. Constitutive promoters from bacteriophage T7

Table 17. Constitutive promoters from bacteriophage SP6

Table 18. Constitutive promoters from yeast

SEO ID NO: 732

BBa K122000

pPGKl . . . ttatctactttttacaacaaatataaaaca 1497 SEO ID NO: 733

BBa K124000

pCYC Yeast Promoter 288 SEO ID NO: 734 acaaacacaaatacacacactaaattaata

BBa K124002 Yeast GPD (TDH3)

681 SEO ID NO: 735 Promoter gtttcgaataaacacacataaacaaacaaa

BBa K319005 yeast mid- length ADH1

720 SEO ID NO: 736 promoter ccaagcatacaatcaactatctcatataca

Yeast CLB 1 promoter

BBa M31201

region, G2/M cell cycle 500 SEO ID NO: 737 accatcaaaggaagctttaatcttctcata

specific

Table 19. Constitutive promoters from miscellaneous eukaryotes

Table 20. Promoters

SEO ID NO: 742

PJ23107+ ggaaaatttttttaaaaaaaaaactttacgg UP element helps recruit RNA polymerase UP element ctagctcagccctaggtattatgctagc

(ggaaaatttttttaaaaaaaaaac)

SEO ID NO: 743

PSYN2311 ggaaaatttttttaaaaaaaaaacTTGA UP element at 5' end; consensus -10 region 9 CAGCTAGCTCAGTCCTTG is TATAAT; the consensus -35 is TTGACA;

GTATAATGCTAGCACGAA the extended -10 region is generally

SEO ID TGNTATAAT (TGGTATAAT in this

sequence)

NO: 744

[0310] In some embodiments, the constitutive promoter is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%

homologous to the sequence of any one of SEQ ID NOs: 598-744.

Ribosome Binding Sites

[0311] In some embodiments, ribosome binding sites are added, switched out or replaced. By testing a few ribosome binding sites, expression levels can be fine-tuned to the desired level. Table A and Table B lists a number RBS which are suitable for

prokaryotic expression and can be used to achieve the desired expression levels (See, e.g., Registry of standard biological parts).

Table 21. Selected Ribosome Binding Sites

BBa_J61107 TCTAGAGAAAGAAGAGACTCACTAGATG 58

BBa_J61108 TCTAGAGAAAGACGAGATATACTAGATG 59

BBa_J61109 TCTAGAGAAAGACTGGAGACACTAGATG 60

BBa_J61110 TCTAGAGAAAGAGGCGAATTACTAGATG 61

BBa_J61111 TCTAGAGAAAGAGGCGATACACTAGATG 62

BBa_J61112 TCTAGAGAAAGAGGTGACATACTAGATG 63

BBa_J61113 TCTAGAGAAAGAGTGGAAAAACTAGATG 64

BBa_J61114 TCTAGAGAAAGATGAGAAGAACTAGATG 65

BBa_J61115 TCTAGAGAAAGAAGGGATACACTAGATG 66

BBa_J61116 TCTAGAGAAAGACATGAGGCACTAGATG 67

BBa_J61117 TCTAGAGAAAGACATGAGTTACTAGATG 68

BBa_J61118 TCTAGAGAAAGAGACGAATCACTAGATG 69

BBa_J61119 TCTAGAGAAAGATTTGATATACTAGATG 70

BBa_J61120 TCTAGAGAAAGACGCGAGAAACTAGATG 1038

BBa_J61121 TCTAGAGAAAGAGACGAGTCACTAGATG 1039

BBa_J61122 TCTAGAGAAAGAGAGGAGCCACTAGATG 1040

BBa_J61123 TCTAGAGAAAGAGATGACTAACTAGATG 1041

BBa_J61124 TCTAGAGAAAGAGCCGACATACTAGATG 1042

BBa_J61125 TCTAGAGAAAGAGCCGAGTTACTAGATG 1043

BBa_J61126 TCTAGAGAAAGAGGTGACTCACTAGATG 1044

BBa_J61127 TCTAGAGAAAGAGTGGAACTACTAGATG 1045

BBa_J61128 TCTAGAGAAAGATAGGACTCACTAGATG 1046

BBa_J61129 TCTAGAGAAAGATTGGACGTACTAGATG 1047

BBa_J61130 TCTAGAGAAAGAAACGACATACTAGATG 1048

BBa_J61131 TCTAGAGAAAGAACCGAATTACTAGATG 1049

BBa_J61132 TCTAGAGAAAGACAGGATTAACTAGATG 1050

BBa_J61133 TCTAGAGAAAGACCCGAGACACTAGATG 1051

BBa_J61134 TCTAGAGAAAGACCGGAAATACTAGATG 1052

BBa_J61135 TCTAGAGAAAGACCGGAGACACTAGATG 1053

BBa_J61136 TCTAGAGAAAGAGCTGAGCAACTAGATG 1054

BBa_J61137 TCTAGAGAAAGAGTAGATCAACTAGATG 1055

BBa_J61138 TCTAGAGAAAGATATGAATAACTAGATG 1056

BBa_J61139 TCTAGAGAAAGATTAGAGTCACTAGATG 1057

Table 22. Selected Ribosome Binding Sites Identifier Sequence³ SEQ ID NO

BBa_B0029 TCTAGAGTTCACACAGGAAACCTACTAGATG 1058

BBa_B0030 TCTAGAGATTAAAGAGGAGAAATACTAGATG 1059

BBa_B0031 TCTAGAGTCACACAGGAAACCTACTAGATG 1060

BBa_B0032 TCTAGAGTCACACAGGAAAGTACTAGATG 1061

BBa_B0033 TCTAGAGTCACACAGGACTACTAGATG 1062

BBa_B0034 TCTAGAGAAAGAGGAGAAATACTAGATG 1063

BBa_B0035 TCTAGAGATTAAAGAGGAGAATACTAGATG 1064

BBa_B0064 TCTAGAGAAAGAGGGGAAATACTAGATG 1065

Induction of Payloads During Strain Culture

[0312] In some embodiments, it is desirable to pre-induce payload or protein of interest expression and/or payload activity prior to administration. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term "bacterial culture" or bacterial cell culture" or "culture" refers to bacterial cells or

microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term "fermentation" refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like.

[0313] Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium. In some embodiments, phenylalanine is added to the media, e.g., to boost cell health. Without wishing to be bound by theory, addition of phenylalanine to the medium may prevent bacteria from catabolizing endogenously produced phenylalanine required for cell growth.

[0314] In some embodiments, the one or more protein(s) of interest and are directly or indirectly induced, while the strains are grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is "wasted", in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut and/or conditions of the tumor

microenvironment (e.g., low oxygen, or in the presence of gut metabolites).

[0315] In one embodiment, expression of one or more payload(s), is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s) is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s) is driven from the one or more different promoters.

[0316] In some embodiments, the strains are administered without any pre- induction protocols during strain growth prior to in vivo administration.

[0317] Anaerobic induction

[0318] In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^A8 to 1X10^A11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s) and /or transporters under the control of one or more FNR promoters.

[0319] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions.

[0320] In one embodiment, expression of two or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s) under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under anaerobic or low oxygen conditions.

[0321] Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut and/or conditions of the tumor microenvironment.

[0322] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) and/or transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

[0323] In one embodiment, expression of one or more Payload is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[0324] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions. [0325] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) and/or transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload and or transporter sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Aerobic induction

[0326] In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g. , ODs within the range of 0.1 to 10, indicating a certain density e.g. , ranging from 1X10^A8 to 1X10^A11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours.

[0327] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

[0328] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions.

[0329] In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[0330] In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

[0331] In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s)and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions.

[0332] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions.

[0333] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments, two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline. [0334] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a

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

[0335] In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment.

[0336] In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[0337] In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment.

[0338] In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene payload and/or transporter sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment.

[0339] As evident from the above non-limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with thermoregulation. Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions.

Microaerobic Induction

[0340] In some embodiments, viability, growth, and activity are optimized by pre-inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to "strike a balance" between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s) and/or transporter(s) are driven by a anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown {e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^A8 to 1X10^A11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for approximately 3 to 5 hours.

[0341] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions. [0342] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions.

[0343] Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut and/or conditions of the tumor microenvironment.

[0344] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.

[0345] In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

[0346] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions.

[0347] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g. , an anaerobic/low oxygen promoter or microaerobic promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise pay load under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Induction of Strains using Phasing, Pulsing and/or Cycling

[0348] In some embodiments, cycling, phasing, or pulsing techniques are emplyed during cell growth, expansion, recovery, purification, fermentation, and/or manufacture to efficienty induce and grow the strains prior to in vivo administration. This method is used to "strike a balance" between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g., for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g. , ODs within the range of 0.1 to 10, indicating a certain density e.g. , ranging from 1X10^A8 to 1X10^A11, and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g. , reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle.

[0349] In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non-liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g., induction of FNR promoters). In one embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed.

[0350] In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non- limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g. , for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1X10^A8 to 1X10^A11. Then the chemical inducer, e.g., arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed.

[0351] In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload. For example, lowering the temperature during culture may improve the proper folding of the payload. In such instances, cells are first grown at a temperature optimal for growth (e.g. , 37 C). In some embodiments, the cells are then induced, e.g. , by a chemical inducer, to express the payload. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload.

[0352] In some embodiments, payload(s) are under the control of different inducible promoters, for example two different chemical inducers. In other

embodiments, the payload is induced under low oxygen conditions or microaerobic conditions and a second payload is induced by a chemical inducer.

[0353] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques.

[0354] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques.

[0355] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) and/or transporter gene sequence(s) and /or transcriptional regulator gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some

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

[0356] Any of the strains described herein can be grown through the

employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. [0357] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques.

[0358] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g. , an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. Any of the strains described in these embodiments, may be induced through the employment of phasing or cycling or pulsing or spiking techniques.

Aerobic induction of the FNR promoter

[0359] FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, an oxygen bypass system shown and described in FIG. 85A is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of the protein of interest {e.g., one or more anti-cancer, satiety, gut barrier enhancer, immune modulatory and/or neuromodulatory effector(s) described herein) by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of the protein of interest. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the protein of interest, e.g., one or more anti-cancer, satiety, gut barrier enhancer, immune modulatory and/or neuromodulatory effector(s) described herein.

[0360] In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments, described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non- limiting example, a protein of interest and FNRS24Y can in some embodiments, be induced simultaneously, e.g. , from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest (e.g., one or more anti-cancer, satiety, gut barrier enhancer, immune modulatory and/or neuromodulatory effector(s) described herein) are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter.

[0361] In some embodiments, a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

Generation of Bacterial Strains with Enhanced Ability to Transport Biomolecules

[0362] Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g. , temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

[0363] This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite.

[0364] For example, if the bio synthetic pathway for producing an amino acid is disrupted a strain capable of high- affinity capture of said amino acid can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the amino acid at regular intervals. Over time, cells that are most competitive for the amino acid - at growth-limiting concentrations - will come to dominate the population. These strains will likely have mutations in their amino acid-transporters resulting in increased ability to import the essential and limiting amino acid.

[0365] Similarly, by using an auxotroph that cannot use an upstream metabolite to form an amino acid, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways.

[0366] In the previous examples, a metabolite innate to the microbe was made essential via mutational auxotrophy and selection was applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate. Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

[0367] Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

[0368] Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations "screened" throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 10 11 2 CCD 1. This rate can be accelerated by the addition of chemical mutagens to the cultures - such as N-methyl-N-nitro-N-nitrosoguanidine (NTG) - which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

[0369] At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvo luted from the evolved strain by

reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. 0. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

[0370] These methods were used to generate E.Coli Nissle mutants that consume kynurenine and over-produce tryptophan as described elsewhere herein. Tryptophan

[0371] 1- Tryptophan (TRP) is one of the nine essential amino acids and is the least abundant of all 21 dietary amino acids in human beings. Dietary TRP is transported from the digestive tract through the portal vein to the liver where it is used for the synthesis of proteins. The distinguishing structural characteristic of TRP is that it contains an indole functional group. Apart from protein synthesis, TRP is used in the generation of products such as serotonin, melatonin, tryptamine, and the products of the kynurenine pathway (KP, collectively called the kynurenines). TRP and its catabolites have well characterized immunosuppressive and disease tolerance functions, and contribute to immune privileged sites such as eyes, brain, placenta, and testes. The kynurenine pathway represents >95% of TRP-catabolizing pathways and is now established as a key regulator of innate and adaptive immunity through its involvement in cancer, autoimmunity, and infection.

[0372] Several KP Pathway metabolites, most notably kynurenine, have been shown to be activating ligands for the arylcarbon receptor (AhR; also known as dioxin receptor). Kynurenine (KYN) was initially shown in the cancer setting as an

endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival.

[0373] In the gut, e kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism. Tryptophan may be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell responses and promotion of Treg cells.

[0374] More recently, additional tryptophan metabolites, collectively termed "indoles", herein, also have been shown to function as AhR agonists. The metabolites include for example, indole-3 aldehyde, indole-3 acetate, indole-3 propionic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc., and tryptamine (are, see e.g., Table 23 and FIG 6A and Fig. 6B) and elsewhere herein, and Lama et al., Nat Med. 2016 Jun;22(6):598-605; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands). The majority of these metabolites are generated by the microbiota; some are generated by the human host and/or taken up from the diet. [0375] Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in addition to kynurenine, other tryptophan metabolites, e.g., indoles (described herein, tryptamine, and kynurenic acide (KYNA) have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF- KB subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR- ARNT associated transcription of IL-6, which induced autocrine activation of IDOl via STAT3. This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer, supporting the idea that IDO/kynurenine-mediated immunosuppression enables the immune escape of tumor cells.

[0376] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of tryptophan levels is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0377] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of tryptophan levels is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the

embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of tryptophan levels is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of tryptophan levels is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0378] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of tryptophan levels may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of tryptophan levels are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0379] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the production of a short chain fatty acid.

[0380] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0381] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the secretion or display of an anti-PD- Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of tryptophan levels further comprise one or more gene sequences for the catabolism of adenosine.

[0382] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0383] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0384] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Kynurenine Pathway

Kynurenine, IDQ, and TDQ

[0385] The rate-limiting conversion of tryptophan to kynurenine (KYN) may be mediated by either of two forms of indoleamine 2, 3-dioxygenase, IDOl expressed ubiquitously, ID02 expressed in kidneys, epididymis, testis, and liver or by tryptophan 2,3-dioxygenase (TDO) expressed in the liver and brain.

[0386] TDO is essential for homeostasis of TRP concentrations and has a lower affinity to TRP than IDOl. Its expression is activated mainly by increased plasma TRP concentrations but can also be activated by glucocorticoids and glucagon.

[0387] The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut and Sci Transl Med. 2013 July 10; 5(193): 193ra91). In some embodiments, the genetically engineered bacteria comprise one or more heterologous bacterially derived genes from Enterobacteriaceae, e.g. whose gene products catalyze the conversion of TRP:KYN.

[0388] In one embodiment, the genetically engineered bacteria comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise a gene or gene cassette for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase ant i- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions. In some embodiments, the genetically engineered bacteria secrete an enzyme which produces kynurenine.

[0389] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenine is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0390] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenine is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenine is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenine is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0391] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenine may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenine are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0392] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the production of a short chain fatty acid.

[0393] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0394] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenine further comprise one or more gene sequences for the catabolism of adenosine.

[0395] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0396] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0397] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Post-kynurenine metabolism

[0398] As shown in FIG. 2, kynurenine is further metabolized along the two distinct routes competing for kynurenine as a substrate: (a) KYN, kynurenic acid (KYNA) pathway; and (b) KYN, nicotinamide adenine dinucleotide (NAD) pathway. Kynurenic Acid, Xanthurenic Acid, Anthranillic Acid

[0399] Kynurenine is further metabolized along the two distinct routes competing for KYN as a substrate: (a) KYN, kynurenic acid (KYNA) pathway; and (b) KYN, nicotinamide adenine dinucleotide (NAD) pathway

[0400] Along one arm, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA).

[0401] KYNA is generated by kynurenine aminotransferases (KAT I, II, III), e.g., in astrocytes in the brain and can also bind AHR and GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-aspartate (NMD A) -receptors. Elevated levels of KYNA have previously been observed in patients with schizophrenia, both in the cerebrospinal fluid (CSF) and postmortem prefrontal cortex.

[0402] The major nerve supply to the gut is also activated the activation of NMD A glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003).

[0403] KYNA also has signaling functions through activation of its recently identified receptor, GPR35. GPR35 is predominantly detected in immune cells in the gastrointestinal tract, and might be involved in nociceptive perception. KYNA might have an ant i- inflammatory effect by inhibition of lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha secretion in peripheral blood mononuclear cells.

[0404] Additionally, KYNA has been found to be generated by macrophages. Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, presumably due to chronic stress or the low-grade inflammation that are prominent risk factors for diabetes.

Thermochemical and kinetic data show that KYNA and XA are the best free-radical scavengers from the eight tested TRP metabolites, suggesting that the production is a regulatory mechanism to attenuate damage by the inflammation- induced production of reactive oxygen species.

[0405] The genetically engineered bacteria may comprise any suitable gene for producing kynurenic acid. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid, e.g., from kynurenine through a circuit comprising gene(s) or gene sequence(s) compring kynurenine-oxoglutarate

transaminase or an equivalent thereof.

[0406] In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions. IN some embodiments, the genetically engineered bacteria secrete an enzyme for the production of kynurenic acid.

The KYN-nicotinamide adenine dinucleotide pathway

[0407] The major enzymes of the KYN-NAD pathway are KYN-3- monooxygenase and kynureninase. Among more than 30 intermediate metabolites (collectively named "kynurenines") are NMDA agonists (quinolinic and picolinic acids) and free radical generators (3-hydroxykynurenine and 3-hydroxyanthranilic acids). One of the major metabolites of this pathway, 3- hydroxykynurenine (3-HK), is a potential neurotoxin involved in several neurodegenerative disorders. The other metabolite, xanthurenic acid, reacts with insulin with formation of a complex antigenetically indistinguishable from insulin. Quinolinic acid (a glutamate receptor agonist) and picolinic acids exert anxiogenic (anxiety causing) effects in animal models, and play a neurotoxic role. Quinolinic and picolinic acids stimulate inducible nitric oxide synthase (iNOS and together with 3 -hydroxykynurenine and 3 -hydroxyanthranilic acids might increase lipid peroxidation, and trigger an arachidonic acid cascade resulting in the increased production of inflammatory factors.

[0408] Anthranilic and xanthurenic acid can act as antioxidants in certain chemical environments. Patients suffering from neurological disorders as Huntington's disease or brain injury often showed decreased levels of xanthurenic acid combined with increased levels of anthranilic acid (AA). However, the biological importance of the 3 -hydroxyanthranilic acid (3-HA) to AA ratio as either neurotoxic or

neuroprotective mechanism is still discussed.

[0409] Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of a number of diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning tryptophan and tryptophan metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites, and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.

[0410] In cells of the nervous system KYNA, anthranilic acid (AA), 3- hydroxyanthranilic acid (3-HA), or QUIN modulate neurological functions. KYNA acts as an antagonist of the glutamate receptor and is therefore described as a

neuroprotective metabolite, whereas QUIN, an agonist of the glutamate receptor, mediates neurotoxic effects. In some embodiments, the genetically engineered bacteria comprise any of the TRP/KP metabolism modulating cassettes described herein, and can beneficically and context-dependently influence the ratios of various TP metabolites in the brain. [0411] In certain embodiments, the genetically engineered bacteria comprise one or more genes(s) or gene cassettes, which can synthesize tryptophan and/or one or more of its metabolites, thereby modulating local and/or systemic concentrations and or ratios of tryptophan and/or one or more of its metabolites.

[0412] In some embodiments, the genetically engineered bacteria modulate the inflammatory status, influence immunosuppression, disease tolerance, or neurological status.

[0413] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0414] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0415] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0416] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the production of a short chain fatty acid.

[0417] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of an antiinflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0418] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of the levels of one or more post-kynurenine metabolites described herein further comprise one or more gene sequences for the catabolism of adenosine.

[0419] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0420] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an anti-inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL- 27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0421] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety. Other Indole Tryptophan Metabolites

[0422] In addition to kynurenine and KYNA, numerous compounds have been proposed as endogenous AHR ligands, many of which are generated through pathways involved in the metabolism of tryptophan and indole (Bittinger et al., 2003; Chung and Gadupudi, 2011) A large number of metabolites generated through the tryptophan indole pathway are generated by microbiota in the gut. For example, bacteria take up tryptophan, which can be converted to mono-substituted indole compounds, such as indole acetic acid (IAA) and tryptamine, and other compounds, which have been found to activate the AHR (Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles; Nature Scientific Reoports 5: 12689).

[0423] In the gastronintestinal tract, diet derived and bacterially AhR ligands promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs (Spits et al., 2013, Zelante et al., Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22; Immunity 39, 372-385, August 22, 2013). AHR is essential for IL-22-production in the intestinal lamina propria (Lee et al., Nature Immunology 13, 144-151 (2012); AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch).

[0424] Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of IL-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.

[0425] Table 8 lists exemplary tryptophan metabolites which have been shown to bind to AhR and which can be produced by the genetically engineered bacteria of the disclosure.

Table 23. Indole Tryptophan Metabolites

Dietary 3.3'-Diindolylmethane (DIM)

Dietary 2- (indo 1- 3 - ylmethy 1) -3.3' -diindo ly lmethane (Ltr- 1 )

Dietary Indolo(3,2-b)carbazole (ICZ)

Dietary 2-(rH-indole-3'-carbony)-thiazole-4-carboxylic acid methyl ester (ITE)

Microbial Indole

Microbial Indole- 3 -acetic acid (IAA)

Microbial Indole-3-aldehyde (IAId)

Microbial Tryptamine

Microbial 3 -methyl- indole (Skatole)

Yeast Tryptanthrin

Microbial/Host Indigo

Metabolism

Microbial/Host Indirubin

Metabolism

Microbial/Host Indoxyl-3-sulfate (I3S)

Metabolism

Host Kynurenine (Kyn)

Metabolism

Host Kynurenic acid (KA)

Metabolism

Host Xanthurenic acid

Metabolism

Host Cinnabarinic acid (CA)

Metabolism

UV-Light 6-formylindolo(3,2-b)carbazole (FICZ)

Oxidation

Microbial

metabolism

[0426] In addition, some indole metabolites may exert their effect through Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function. PXR-deficient (Nrli2-/-) mice showed a distinctly "leaky"gut physiology coupled with upregulation of the To 11- like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). In particular, indole 3-propionic acid (IP A), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo. [0427] As a result of PXR agonism, indole metabolite levels e.g., produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. In other words, low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinal barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health.

[0428] Although microbial degradation of tryptophan to indole-3-propionate has been shown in a numver of microorganisms (see, e.g., Elsden et al., The end products of the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr l;107(3):283-8), to date, the bacterial entire biosynthetic pathway from tryptophan to IPA is unknown. In Clostridium sporogenes, tryptophan is catabolized via indole-3- pyruvate, indole-3-lactate, and indole- 3 -aery late to indole-3-propionate (O'Neill and DeMoss, Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys. 1968 Sep 20;127(l):361-9). Two enzymes that have been purified from C. sporogenes are tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss, Indolelactate dehydrogenase from Clostridium sporogenes, Can J Microbiol. 1968 Apr;14(4):429-35). Lactococcus lactis, catabolizes tryptophan by an

aminotransferase to indole-3-pyruvate. In Lactobacillus casei and Lactobacillus helveticus tryptophan is also catabolized to indole-3-lactate through successive transamination and dehydrogenation (see, e.g., Tryptophan catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-2077, and references therein).

[0429] L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g., Clostridium sporogenes or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indol- 3yl)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g., Clostridium sporogenes orLactobaciUus casei) converts (indol-3yl) pyruvate and NADH and H+ to indole- 3 lactate and NAD+.

[0430] In some embodiments, the engineered bacteria encode one or more enzymes selected from tryptophan transaminase (e.g., from C. sporogenes) and/or indole- 3 -lactate dehydrogenase (e.g., from C. sporogenes), and/or indole-3-pyruvate aminotransferase (e.g., from Lactococcus lactis). In other embodiments, such enzymes encoded by the bacteria are from Lactobacillus casei and/or Lactobacillus helveticus. [0431] In other embodiments, IPA producing circuits comprise enzymes depicted and described in FIG. 6A and FIG. 6B and elsewhere herein.

Methoxyindole pathway, Serotonin and Melatonin

[0432] The methoxyindole pathway leads to formation of serotonin (5-HT) and melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine synthesized in a two-step enzymatic reaction: First, enzymes encoded by one of two tryptophan hydroxylase genes (Tphl or Tph2) catalyze the rate-limiting conversion of tryptophan to 5-hydroxytryptophan (5-HTP). Tphl 2 is active in the brain and Tph2 is active in the periphery. Subsequently, 5-HTP undergoes decarboxylation to serotonin. Serotonin metabolism is independently regulated in the brain and periphery because the blood- brain barrier partitions bioavailability.

[0433] The majority (95%-98%) of total body serotonin is found in the gut (Berger et al, 2009). Peripheral serotonin acts autonomously on many cells, tissues, and organs, including the cardiovascular, gastrointestinal, hematopoietic, and immune systems as well as bone, liver, and placenta (Amireault et al., 2013). Serotonin functions as a ligand for any of 15 membrane-bound mostly G protein-coupled serotonin receptors (5-HTRs) that are involved in various signal transduction pathways in both CNS and periphery. Intestinal serotonin is released by enterochromaffin cells and neurons and is regulated via the serotonin re-uptake transporter (SERT). The SERT is located on epithelial cells and neurons in the intestine. Gut microbiota are interconnected with serotonin signaling and are for example capable of increasing serotonin levels through host serotonin production (Jano et al., Cell. 2015 Apr 9;161(2):264-76. doi:

10.1016/j.cell.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis). These studies found that GI 5-HT synthesis, which modulates circulating levels, is driven by microbial metabolites such as SCFAs or tryptophan- derived indole metabolites.

[0434] Modulation of tryptophan metabolism, especially serotonin synthesis is considered a novel potential strategy the treatment of gastrointestinal (GI) disorders. For example, diarrhea-predominant irritable bowel disorder (IBD) is associated with elevated serotonin, while constipation-predominant IBD) is associated with decreased levels of serotonin in the colon mucosa. [0435] In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the gut, e.g. , decrease or increase serotonin levels in the gut. In certain embodiments, the genetically engineered bacteria influence serotonin synthesis, release, and/or degradation. In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to improve gut barrier function, modulate the inflammatory status or otherwise ameliorate symptoms of a metabolic disease and/or an gastrointestinal disorder or inflammatory bowel disorder. In some embodiments, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In some embodiments, the genetically engineered bacteria release serotonin into the environment, e.g., the gut. In some embodiments, the genetically engineered modulate or influence serotonin levels produced by the host. In some embodiments, the genetically engineered bacteria counteract microbiota which are responsible for altered serotonin function in many GI diseases.

[0436] In some embodiments, the genetically engineered bacteria comprise tryptophan hydroxylase (TpH (land/or2)) and/or 1- amino acid decarboxylase, e.g. for the treatment of constipation-associated IBD. In some embodiments, the genetically engineered bacteria comprise cassettes which allow trptophan uptake and catalysis, reducing trptophan availability for serotonin synthesis (serotonin depletion). In some embodiments, the genetically engineered bacteria comprise cassettes which promote serotonin uptake from the environment, e.g. , the gut, and serotonin catalysis.

[0437] The Tph2-dependent serotoninergic system acts solely at specific sites in the brain, which accounts for 2%-5% of total body serotonin. In the brain, serotonin modulates mood, anxiety, appetite, and potentially cognitive performance. In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the brain.

[0438] It has been shown that indole metabolites promote serotonin synthesis and release. In some embodiments, the genetically engineered bacteria comprise gene sequences encoding one or more of any of the indole production enzymes and cassettes described herein, e.g., to promote serotonin synthesis and release. In some

embodiments, the genetically engineered bacteria produce indole and/or SCFA metabolite to promote serotonin release, e.g., for the treatment, prevention, and/or management of IBD, IBS and/or CNS related disorders. [0439] Additionally, serotonin also functions a substrate for melatonin biosynthesis. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle. Melatonin is a well-known lipophylic hormone produced at night by pineal gland and after feeding with tryptophan containing protein or tryptophan itself by neuroendocrine cells of the digestive system. It acts through high-affinity Gprotein-coupled membrane receptors through endocrine, paracrine or neurocrine pathway to protect the mucosa of the upper gastrointestinal tract from various irritants and ulcerogens.

[0440] The rate-limiting step of melatonin biosynthesis is 5-HT-N-acetylation resulting in the formation of N-acetyl-serotonin (NAS) with subsequent Omethylation into 5-methoxy-N-acetyltryptamine (melatonin). The deficient production of 5-HT, NAS, and melatonin contribute to depressed mood, disturbances of sleep and circadian rhythms.

[0441] In bacteria, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimic acid pathway. In these cells, synthesis starts with d-erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments, the genetically engineered bacteria comprise an endogenous or exogenous cassette for the production of melatonin. As a non-limiting example, the cassette is described in Bochkov, Denis V.; Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011). "Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources". Journal of Chemical Biology 5 (1): 5-17. doi: 10.1007/sl2154-011-0064-8.

[0442] In a non-limiting example, genetically engineered bacteria convert tryptophan and/or serotonin to melatonin by, e.g., , tryptophan hydroxylase (TPH), hydroxyl-O-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic - amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial equivalents.

[0443] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0444] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels is operably linked to a RBS, enhancer or other regulatory sequence. In some

embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0445] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome. [0446] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the production of a short chain fatty acid.

[0447] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the production of butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0448] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the modulation of serotonin and/or melatonin levels further comprise one or more gene sequences for the catabolism of adenosine.

[0449] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0450] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0451] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety. Tryptophan and Tryptophan Metabolite Circuits

Decreasing Exogenous Tryptophan

[0452] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan and/or the level of a tryptophan metabolite. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. Tryptophan transporters may be expressed or modified in the recombinant bacteria described herein in order to enhance tryptophan transport into the cell. Specifically, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a tryptophan transporter which may be used to import tryptophan into the bacteria.

[0453] The uptake of tryptophan into bacterial cells is mediated by proteins well known to those of skill in the art. For example, three different tryptophan transporters, distinguishable on the basis of their affinity for tryptophan have been identified in E. coli (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake in bacteria. High affinity permease, Mtr, is negatively regulated by the trp repressor and positively regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17 and Heatwole et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is negatively regulated by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93).

[0454] In one embodiment, the at least one gene encoding a tryptophan transporter is a gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the bacterial cell described herein has been genetically engineered to comprise at least one heterologous gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli mtr gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli aroP gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli tnaB gene. [0455] In some embodiments, the tryptophan transporter is encoded by a tryptophan transporter gene derived from a bacterial genus or species, including but not limited to, Escherichia, Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or Corynebacterium glutamicum. In some embodiments, the bacterial species is

Escherichia coli. In some embodiments, the bacterial species is Escherichia coli strain Nissle.

[0456] Assays for testing the activity of a tryptophan transporter, a functional variant of a tryptophan transporter, or a functional fragment of transporter of tryptophan are well known to one of ordinary skill in the art. For example, import of tryptophan may be determined using the methods as described in Shang et al. (2013) J. Bacteriol. 195:5334-42, the entire contents of each of which are expressly incorporated by reference herein.

[0457] In one embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another

embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the

recombinant bacterial cells described herein, the bacterial cells import two-fold more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six- fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty- fold, or fifty- fold, more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.

[0458] In addition to the tryptophan uptake transporters, in some embodiments, the genetically engineered bacteria further comprise a circuit for the production of tryptophan metabolites, as described herein, e.g., for the production of kynurenine, kynurenine metabolites, or indole tryptophan metabolites as shown in Table 23. [0459] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

[0460] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan, e.g., in combination with the production of indole metabolites, through expression of gene(s) and gene cassette(s) described herein.

[0461] Increasing Kynurenine

[0462] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine.

[0463] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise on or more gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.

[0464] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenine from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 16E and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ID01(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDOl from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S.

cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine— oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene

sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.

[0465] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.

[0466] In any of these embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 19A and/or FIG. 19B FIG. 20, FIG. 21(A-C) and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported.

Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0467] The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some

embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with disorders, such as liver damage, metabolic disease, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose .

[0468] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some

embodiments, the enzymes are derived from the species listed in table S7 of Vujkovic- Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

[0469] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynurenineis operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. [0470] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynurenineis operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynurenineis operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynurenineis modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0471] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninemay be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynurenineare present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0472] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the production of a short chain fatty acid.

[0473] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0474] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM- CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan and/or production of kynureninefurther comprise one or more gene sequences for the catabolism of adenosine. [0475] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0476] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0477] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Increasing Tryptophan and Degrading kynurenine

[0478] In some embodiments, the genetically engineered microorganisms of the present disclosure are capable of producing tryptophan. Exemplary circuits for the production of tryptophan are shown in FIG. 1A, FIG. IB, FIG 1C, and FIG. ID.

[0479] In some embodiments, the genetically engineered bacteria that produce tryptophan comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon. In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of E. coli. (Yanofsky, RNA (2007), 13: 1141-1154). In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of B. subtilis. (Yanofsky, RNA (2007), 13: 1141-1154). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC- F, trypB, and trpA genes from E. Coli. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis.

[0480] Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, chorismate. Thus, in some embodiments, the genetically engineered bacteria optionally comprise sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway and one or more gene sequences encoding one or more enzymes of the chorismate biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes.

[0481] In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 10). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a feedback resistant SerA gene (Table 10). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type SerA gene (Table 10). Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD+ to NADH. As part of Tryptophan biosynthesis, E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved (see, e.g., FIG. 22D.

[0482] In any of these embodiments, AroG is optionally replaced with feedback resistant versions to improve tryptophan production (Table 25). In any of these embodiments, TrpE is optionally replaced with feedback resistant versions to improve tryptophan production (Table 25).

[0483] In any of these embodiments, AroG (2-dehydro-3- deoxyphosphoheptonate aldolase) and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 25).

[0484] In any of these embodiments, the tryptophan repressor (TrpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function.

[0485] In any of these embodiments, the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 25).

[0486] The inner membrane protein YddG of Escherichia coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbial Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over- express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

[0487] In some embodiments, the genetically engineered bacteria comprise a mechanism for metabolizing or degrading kynurenine, which, in some embodiments, also results in the increased production of tryptophan. In some embodiments, the genetically engineered bacteria modulate the TRP:KYN ratio or the KYN:TRP ratio in the extracellular environment. In some embodiments, the genetically engineered bacteria increase the TRP:KYN ratio or the KYN:TRP ratio. In some embodiments, the genetically engineered bacteria reduce the TRP:KYN ratio or the KYN:TRP ratio. In some embodiments, the genetically engineered bacteria comprise sequence encoding the enzyme kynureninase. Kynureninase is produced to metabolize Kynurenine to

Anthranilic acid in the cell. Schwarcz et al., Nature Reviews Neuroscience, 13, 465- 477; 2012; Chen & Guillemin, 2009; 2; 1-19; Intl. J. Tryptophan Res. Exemplary kynureninase sequences are provided herein below in Table 26. In some embodiments, the engineered microbe has a mechanism for importing (transporting) kynurenine from the local environment into the cell. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter.

[0488] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes of the tryptophan biosynthetic pathway and sequence encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon, for example that of E. coli. or B. subtilis, and sequence encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes, for example, from E. Coli and sequence encoding kyureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes, for example from B. subtilis and sequence encoding kyureninase. In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, for example, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. Thus, in some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes, and sequence encoding kyureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes, and sequence encoding kyureninase.

[0489] In some embodiments, the genetically engineered bacteria may optionally have a deletion or mutation in the endogenous trpE, rendering trpE nonfunctional. Accordingly, in one embodiment, the genetically engineered bacteria may comprise one or more gene(s) or gene cassette(s) encoding trpD, trpC, trpA, and trpD and kynureninase (see, e.g. FIG. 1 and FIG. 9). This deletion may prevent tryptophan production through the endogenous chorismate pathway, and may increase the production of tryptophan from kynurenine through kynureninase.

[0490] In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 25) .

[0491] In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 25).

[0492] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function.

[0493] In any of these embodiments, the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 25).

[0494] In some embodiments, the genetically engineered bacteria comprising gene sequence(s) for the production of kynureninase further comprise a mutation and or deletion in TyrB. Tyrosine aminotransferase (TyrB) also known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis and can also catalyze the conversion of kynurenine into kynurenic acid. Such a deletion may prevent kynurenine from being degraded into kynurenic acid (another Ahr agonist) rather than being converted into anthanillic acid by kynureninase.

[0495] In any of these embodiments, the genetically engineered bacterium may further comprise gene sequence for exporting or secreting tryptophan from the cell. Thus, in some embodiments, the engineered bacteria further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG, an aromatic amino acid exporter. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene. In any of these embodiments, the genetically engineered bacterium may further comprise gene sequence for importing or transporting kynurenine into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene. [0496] In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan and/or kynureninase, under the control of a promoter that is activated by low-oxygen conditions, by inflammatory conditions, liver damage, and. or metabolic disease, such as any of the promoters activated by said conditions and described herein. In some embodiments, the genetically engineered bacteria expresses one or more genes for producing tryptophan and/or kynureninase, under the control of a cancer- specific promoter, a tissue- specific promoter, or a constitutive promoter, such as any of the promoters described herein. Table 24 lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure.

Table 24A. Tryptophan Synthesis Cassette Sequences

atcgactcttttacgtacaacctggcagatcagttgcgcagcaatggtcataacgtggtgatttaccgcaaccata ttccggcgcagaccttaattgaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggt gtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttg cctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcttcacggtaaa gcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaaacccgctgccagtggcgcgttatc actcgctggttggcagtaacattccggccggtttaaccatcaacgcccattttaatggcatggtgatggcggtgc gtcacgatgcagatcgcgtttgtggattccagttccatccggaatccattcttactacccagggcgctcgcctgct ggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattctggaaaaa ctgtatcaggcacagacgcttagccaacaagaaagccaccagctgttttcagcggtggtacgtggcgagctga agccggaacaactggcggcggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccgg ggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgtcggt actggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcgggctga aagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctggcggcgttcggtat taatcttgatatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaa gtatcacaccggattccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctg ggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtccggaactggtgctgccgatt gccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggcgggatggatgaagtttc attacacgcgccgacaatcgttgccgaactacatgacggcgaaattaagagctatcaattgaccgctgaagatt ttggcctgacaccctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacac gcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcct gcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtggttccgcttacgaca gagtcaccgcactggcggcacgagggtaaatgatgcaaaccgttttagcgaaaatcgtcgcagacaaggcg atttgggtagaaacccgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga catttttatgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggc gtgatccgtgatgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaatttcagtgctgac tgatgagaaatattttcaggggagctttgatttcctccccatcgtcagccaaatcgccccgcagccgattttatgta aagacttcattatcgatccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcag tactggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagt cagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggcatcaacaaccgcgatct gcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaactggggcacaacgtgacggtaatc agcgaatccggcatcaatacttacgctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcg gcgttgatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcct gacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgcgacat caccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccgttgcagtatgttggcgtgtt ccgcaatcacgatattgccgatgtggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggt aatgaagatcagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaag tgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggga gcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctggcggggggcttaggc gcagataactgcgtggaagcggcacaaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaac cgggtatcaaagacgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaatga caacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgcca gctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaaact atgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatctga agcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcg ccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccgg atgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatgagg cgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatcctta cccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagagaagg tcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatcaac gaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgcacc gttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaatt gaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcact ggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaagg gatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaa agagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagc acgaggggaaatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattc gttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtg ctgacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacact gcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccg accattcccatcggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcga gaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgc gttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctctt acggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccc tcaatcatctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccgg atcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgag caacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgc gcagttaatacgcatggcatggatgaCCGATGGTAGTGTGGGGTCTCCCCATGCG

AGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGT

CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC

TCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGC

GAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAA

CTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCC

TTTTTGCGTGGCCAGTGCCAAGCTTGCATGCGTGC

Tet repressor taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaaggctggctct SEQ ID gcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct NO:72 tctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata atgcattctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtagg ccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa aaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacg ggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagaca t

tetR/tetA cattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtga promoters and actctagaaataattttgtttaactttaagaaggagatatacat

RBS and

leader region

SEQ ID NO

73:

trpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttt

SEQ ID NO: tcaccagttgtgtggggatcgtccggcaacgctgctgctggaatccgcagatatcgacagcaaagatgatttaa 74 aaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaatccaggcgctttcc ggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaa actgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttt tgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcggcggcct gttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaaaatagctgccctgatttctgttt ttatctcgctgaaacgctgatggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctcc gaatgaagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgc cgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgta gtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctcgccgtttctctctgccct gcccgtcaccgctggcagcctattacgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggata atgatttcaccctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgagattt acccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatctcgcccgtaatgac ctggcacgcatttgcacacccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgat gcacctagtctcccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgac gcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcattgtgatccgctc ggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtccttgattctgttccgcagtcg gaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggag acgttcta

trpD atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggtc

SEQ ID NO: ataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcgacgatgagcaatccg 76 gtgctgatgctttctcctggccccggtgtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcg tggcaagctgccaattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtca ggcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaaca aacccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaaccatcaacgccca ttttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccatt cttactacccagggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttt tcagcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcatgaaaattcgcgg tgaacacccgaacgagatcgccggggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccg gattatctgtttgccgatatcgtcggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcg tttgtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcg tcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgctggatgagtta ggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcgatgccggttcgccagcaactgaa aacccgcactctgttcaacgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgttta tagtccggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgca cagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcgaaattaag agctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagcaattggcaggcggaacaccgg aagaaaaccgtgacattttaacacgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcgg cgaatgtcgccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggta ctgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggtaa

trpC atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaaccg

SEQ ID NO: ctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttcagggcgcacgcacggc 78 gtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgc cgccatttataaacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcct ccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaatatcgccagcttgca gccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagtaatgaagaggaactggagcgcgccatt gcattgggggcaaaggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggt gcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgatttgaacgccgc cgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtgggcaagatgctaaagcagcttat gacgcgggcgcgatttacggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgca ggaagtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggaca aagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaatctgcgt gaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaactcttcccgcgcgcgattttca gcacatcgataaatatgtattcgacaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatg gtcaatcgcttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaaccg gctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctcggt tttccagacgctgcgcgcatattaa

trpB atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcg

SEQ ID NO: ccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaa 80 actatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatc tgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcga agcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgcca gcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttc cggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatc cttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagaga aggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatc aacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgc accgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaa attgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagc actggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaa gggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaa aaagagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaa gcacgaggggaaatctga

trpA atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc

SEQ ID NO: ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagtt 82 aggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcg ggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggcc ttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcga ttcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacac ctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcg aagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccg cgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagc cagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa

[0497] In one embodiment, the genetically engineered bacteria comprise a

sequence which has at least about 80% identity with one or more sequences of Table

24A. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 24A. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 24A. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95%

identity with one or more sequences of Table 24A. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of

Table 24A. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 2 A. In another embodiment, the genetically engineered bacteria comprise the sequence of Table 24A. In one embodiment, the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 2 A.

[0498] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a Tryptophan operon. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 71. In another embodiment, the genetically engineered bacteria comprise a Tryptophan operon gene sequence which has at least about 85% identity with SEQ ID NO: 71. In one embodiment, the genetically engineered bacteria comprise a

Tryptophan operon gene sequence which has at least about 90% identity with SEQ ID NO: 71. In one embodiment, the genetically engineered bacteria comprise a

Tryptophan operon gene sequence which has at least about 95% identity with SEQ ID NO: 71. In another embodiment, the Tryptophan operon gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 71. Accordingly, in one embodiment, the genetically engineered bacteria comprise a Tryptophan operon gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 71. In another embodiment, the genetically engineered bacteria comprise the Tryptophan operon gene sequence of SEQ ID NO: 71. In yet another embodiment the genetically engineered bacteria comprise a Tryptophan operon gene sequence which consists of the sequence of SEQ ID NO: 71.

[0499] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpE. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 74. In another embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 85% identity with SEQ ID NO: 74. In one embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 90% identity with SEQ ID NO: 74. In one embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 95% identity with SEQ ID NO: 74. In another embodiment, the TrpE gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 74. Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 74. In another embodiment, the genetically engineered bacteria comprise the TrpE gene sequence of SEQ ID NO: 74. In yet another embodiment the genetically engineered bacteria comprise a TrpE gene sequence which consists of the sequence of SEQ ID NO: 74.

[0500] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpD. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 76. In another embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 85% identity with SEQ ID NO: 76. In one embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 90% identity with SEQ ID NO: 76. In one embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 95% identity with SEQ ID NO: 76. In another embodiment, the TrpD gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 76. Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 76. In another embodiment, the genetically engineered bacteria comprise the TrpD gene sequence of SEQ ID NO: 76. In yet another embodiment the genetically engineered bacteria comprise a TrpD gene sequence which consists of the sequence of SEQ ID NO: 76.

[0501] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpC. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 78. In another embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 85% identity with SEQ ID NO: 78. In one embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 90% identity with SEQ ID NO: 78. In one embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 95% identity with SEQ ID NO: 78. In another embodiment, the TrpC gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 78. Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 78. In another embodiment, the genetically engineered bacteria comprise the TrpC gene sequence of SEQ ID NO: 78. In yet another embodiment the genetically engineered bacteria comprise a TrpC gene sequence which consists of the sequence of SEQ ID NO: 78.

[0502] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpB. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 80. In another embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 85% identity with SEQ ID NO: 80. In one embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 90% identity with SEQ ID NO: 80. In one embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 95% identity with SEQ ID NO: 80. In another embodiment, the TrpB gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 80. Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 80. In another embodiment, the genetically engineered bacteria comprise the TrpB gene sequence of SEQ ID NO: 80. In yet another embodiment the genetically engineered bacteria comprise a TrpB gene sequence which consists of the sequence of SEQ ID NO: 80.

[0503] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpA. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 82. In another embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 85% identity with SEQ ID NO: 82. In one embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 90% identity with SEQ ID NO: 82. In one embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 95% identity with SEQ ID NO: 82. In another embodiment, the TrpA gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 82. Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 82. In another embodiment, the genetically engineered bacteria comprise the TrpA gene sequence of SEQ ID NO: 82. In yet another embodiment the genetically engineered bacteria comprise a TrpA gene sequence which consists of the sequence of SEQ ID NO: 82.

Table 24B. Polypeptide Sequences

ALVSMKIRGEHPNEIAGAAT ALLEN AAPFPRPDYLFADIVGTGGDGSN S INIS T AS AFV A A ACGLKV AKHGNRS VS S KS GS S DLL A AFGINLDMN A DKSRQALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLFNVLGPLI NP AHPPL ALIG V YS PELVLPI AETLRVLG YQR A A V VHS GGMDE VS LH APTIVAELHDGEIKSYQLTAEDFGLTPYHQEQLAGGTPEENRDILTRLL QGKGD AAHE AA V AANV AMLMRLHGHEDLQAN AQT VLE VLRS GS A YDRVTALAARG

TrpC MQT VL AKI V AD KAI WVETRKEQQPL AS FQNE VQPS TRHFYD ALQG A

RT AFILEC KKAS PS KG VIRDDFDP ARI A AI YKH Y AS AIS VLTDE KYFQG

SEQ ID NO: SFDFLPIVSQIAPQPILCKDFIIDPYQIYLARYYQADACLLMLSVLDDEQ 79 YRQL A A V AHS LEMG VLTE VS NEEELERAI ALG AKV VGINNRDLRDLS

IDLNRTRELAPKLGHNVTVISESGINTYAQVRELSHFANGFLIGSALM

AHDDLNAAVRRVLLGENKVCGLTRGQDAKAAYDAGAIYGGLIFVAT

SPRCVNVEQAQEVMAAAPLQYVGVFRNHDIADVADKAKVLSLAAV

QLHGNEDQLYIDNLREALPAHVAIWKALSVGETLPARDFQHIDKYVF

DNGQGGSGQRFDWSLLNGQSLGNVLLAGGLGADNCVEAAQTGCAG

LDFNSAVESQPGIKDARLLASVFQTLRAY

[0504] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpE. In some embodiments, TrpE has at least about 80% identity with SEQ ID NO: 75. In some embodiments, TrpE has at least about 85% identity with one or more of SEQ ID NO: 75. In some embodiments, TrpE has at least about 90% identity with SEQ ID NO: 75. In some embodiments, TrpE has at least about 95% identity with SEQ ID NO: 75. In some embodiments, TrpE has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:75. Accordingly, In some embodiments, TrpE has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with

SEQ ID NO: 75. In another embodiment,TrpE comprises the sequence of SEQ ID NO:

75. In another embodiment, TrpE consists of the sequence of one or more of SEQ ID

NO: 75.

[0505] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpA. In some embodiments, TrpA has at least about 80% identity with SEQ ID NO: 83. In some embodiments, TrpA has at least about 85% identity with one or more of SEQ ID NO: 83. In some embodiments, TrpA has at least about 90% identity with SEQ ID NO: 83. In some embodiments, TrpA has at least about 95% identity with SEQ ID NO: 83. In some embodiments, TrpA has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:83. Accordingly, In some embodiments, TrpA has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 83. In another embodiment,TrpA comprises the sequence of SEQ ID NO: 83. In another embodiment, TrpA consists of the sequence of one or more of SEQ ID NO: 83.

[0506] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpB. In some embodiments, TrpB has at least about 80% identity with SEQ ID NO: 81. In some embodiments, TrpB has at least about 85% identity with one or more of SEQ ID NO: 81. In some embodiments, TrpB has at least about 90% identity with SEQ ID NO: 81. In some embodiments, TrpB has at least about 95% identity with SEQ ID NO: 81. In some embodiments, TrpB has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. Accordingly, In some embodiments, TrpB has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 81. In another embodiment,TrpB comprises the sequence of SEQ ID NO: 81. In another embodiment, TrpB consists of the sequence of one or more of SEQ ID NO: 81.

[0507] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpD. In some embodiments, TrpD has at least about 80% identity with SEQ ID NO: 77. In some embodiments, TrpD has at least about 85% identity with one or more of SEQ ID NO: 77. In some embodiments, TrpD has at least about 90% identity with SEQ ID NO: 77. In some embodiments, TrpD has at least about 95% identity with SEQ ID NO: 77. In some embodiments, TrpD has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:77. Accordingly, In some embodiments, TrpD has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 77. In another embodiment,TrpD comprises the sequence of SEQ ID NO: 77. In another embodiment, TrpD consists of the sequence of one or more of SEQ ID NO: 77.

[0508] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpC. In some embodiments, TrpC has at least about 80% identity with SEQ ID NO: 79. In some embodiments, TrpC has at least about 85% identity with one or more of SEQ ID NO: 79. In some embodiments, TrpC has at least about 90% identity with SEQ ID NO: 79. In some embodiments, TrpC has at least about 95% identity with SEQ ID NO: 79. In some embodiments, TrpC has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:79. Accordingly, In some embodiments, TrpC has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79. In another embodiment,TrpC comprises the sequence of SEQ ID NO: 79. In another embodiment, TrpC consists of the sequence of one or more of SEQ ID NO: 79.

[0509] Table 25 depicts exemplary polypeptide sequences feedback resistant AroG and TrpE. Table 25 also depicts an exemplary TnaA (tryptophanase from E. coli) sequence. IN some embodiments, the sequence is encoded in circuits for tryptophan catabolism to indole; in other embodimetns, the sequence is deleted from the E coli chromosome to increase levels of tryptophan.

Table 25A. Feedback resistant AroG and TrpE and tryptophanase sequences

SerA: 2- MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL oxoglutarate DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT reductase from E. NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE coli Nissle ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE

S LGM Y V YFYDIENKLPLGN ATQ VQHLS DLLNMS D V VS LH VPE

SEQ ID NO: 86 NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK

HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA

QENIGLE V AGKLIKYS DNGS TLS A VNFPE VS LPLHGGRRLMHI

HENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA

DEDVAEKALQAMKAIPGTIRARLLY

SerAfbr: feedback MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL resistant 2- DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT oxoglutarate NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE reductase from E. ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE coli Nissle S LGM YV YFYDIENKLPLGN ATQ VQHLS DLLNMS D V VS LH VPE

NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK

SEQ ID NO: 87 HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA

QENIGLE V AGKLIKYS DNGS TLS A VNFPE VS LPLHGGRRLMHI

AEARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA

DEDVAEKALQAMKAIPGTIRARLLY

TnaA: MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE tryptophanase from DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE E. coli SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM

VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN

SEQ ID NO: 88 FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKVM

YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE

TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT

LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA

QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPA

QALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTI

PRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFT

AKLKEV

[0510] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AroGfbr. In some embodiments, AroGfbr has at least about 80% identity with SEQ ID NO: 84. In some embodiments, AroGfbr has at least about 85% identity with one or more of SEQ ID NO: 84. In some embodiments, AroGfbr has at least about 90% identity with SEQ ID NO: 84. In some embodiments, AroGfbr has at least about 95% identity with SEQ ID NO: 84. In some embodiments, AroGfbr has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:84.

Accordingly, In some embodiments, AroGfbr has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 84. In some embodiments,AroGfbr comprises the sequence of SEQ ID NO: 84. In some embodiments, AroGfbr consists of the sequence of one or more of SEQ ID NO: 84.

[0511] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpEfbr. In some embodiments, TrpEfbr has at least about 80% identity with SEQ ID NO: 85. In some embodiments, TrpEfbr has at least about 85% identity with one or more of SEQ ID NO: 85. In some embodiments, TrpEfbr has at least about 90% identity with SEQ ID NO: 85. In some embodiments, TrpEfbr has at least about 95% identity with SEQ ID NO: 85. In some embodiments, TrpEfbr has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:85.

Accordingly, In some embodiments, TrpEfbr has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 85. In some embodiments,TrpEfbr comprises the sequence of SEQ ID NO: 85. In some embodiments, TrpEfbr consists of the sequence of one or more of SEQ ID NO: 85.

[0512] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding SerA. In some embodiments, SerA has at least about 80% identity with SEQ ID NO: 86. In some embodiments, SerA has at least about 85% identity with one or more of SEQ ID NO: 86. In some embodiments, SerA has at least about 90% identity with SEQ ID NO: 86. In some embodiments, SerA has at least about 95% identity with SEQ ID NO: 86. In some embodiments, SerA has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:86. Accordingly, In some embodiments, SerA has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 86. In some embodiments, SerA comprises the sequence of SEQ ID NO: 86. In some embodiments, SerA consists of the sequence of one or more of SEQ ID NO: 86.

[0513] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding SerAfbr. In some embodiments, SerAfbr has at least about 80% identity with SEQ ID NO: 87. In some embodiments, SerAfbr has at least about 85% identity with one or more of SEQ ID NO: 87. In some embodiments, SerAfbr has at least about 90% identity with SEQ ID NO: 87. In some embodiments, SerAfbr has at least about 95% identity with SEQ ID NO: 87. In some embodiments, SerAfbr has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:87. Accordingly, In some embodiments, SerAfbr has at least about 80%, 81%, 82%, 83%, 87%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 87. In some embodiments, SerAfbr comprises the sequence of SEQ ID NO: 87. In some embodiments, SerAfbr consists of the sequence of one or more of SEQ ID NO: 87.

[0514] In one embodiment, TnaA is mutated or deleted.

[0515] Table 25B lists exemplary polynucleotide sequences useful for

tryptophan production.

Table 25B. Sequences Useful for Tryptophan Production

Description Sequence

fbrTrpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgc gctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattcgcagatatcgacagcaaa SEQ ID NO gatgatttaaaaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaa tccaggcgctttccggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtgg aaaatgaacaatcaccaaactgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacg cccgcttatgctccctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaaga acgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttaccgcaact gtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgaccatcagaa aaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgctcg cctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtttccgtgccgcata tgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcg cgccggagaaattttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcc tattacgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccctgtttg gcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgcc ggaacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgcatcgaac tggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatctcgcccgtaatgacctg gcacgcatttgcacacccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtga tgcacctagtctcccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcct gtatgaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaa ggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctg cattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtcct tgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgcc accgcgcatcatgcacaggagacgttcta

fbrAroG atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctgga aaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct

SEQ ID NO: 167 gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgcggctaaag agtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctggaaatcgtgatgcgcgtct attttgaaaagccgcgtactacggtgggctggaaagggctgattaacgatccgcatatggataacagctt ccagatcaacgacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagc ggcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggc gcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgcactgcttcct gtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacggcgattgccatatcattctg cgcggcggtaaagagcctaactacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaa gcaggcctgccagcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcag atggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgtgatggtgg aaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagca tcaccgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgcagtaaaagc gcgtcgcgggtaa

[0516] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 25B. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 25B. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 25B. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 25B. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 25B. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 25B. In another

embodiment, the genetically engineered bacteria comprise the sequence of Table 25B embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 25B.

[0517] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding TrpEfbr. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 185. In another embodiment, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 85% identity with SEQ ID NO: 185. In some embodiments, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 90% identity with SEQ ID NO: 185. In some embodiments, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 95% identity with SEQ ID NO: 185. In another embodiment, the TrpEfbr gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 185. Accordingly, In some embodiments, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 185. In another embodiment, the genetically engineered bacteria comprise the TrpEfbr gene sequence of SEQ ID NO: 185. In yet another embodiment the genetically engineered bacteria comprise a TrpEfbr gene sequence which consists of the sequence of SEQ ID NO: 185.

[0518] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding feedback resistant AroG. In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 80% identity with SEQ ID NO: 167. In another embodiment, the genetically engineered bacteria comprise a AroGfbr sequence which has at least about 85% identity with SEQ ID NO: 167. In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 90% identity with SEQ ID NO: 167. In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 95% identity with SEQ ID NO: 167. In another embodiment, the a AroGfbr gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 167. Accordingly, In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 167. In another embodiment, the genetically engineered bacteria comprise the a AroGfbr gene sequence of SEQ ID NO: 167. In yet another embodiment the genetically engineered bacteria comprise a AroGfbr gene sequence which consists of the sequence of SEQ ID NO: 167.

[0519] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding SerA. In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 80% identity with SEQ ID NO: 169. In another embodiment, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 85% identity with SEQ ID NO: 169. In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 90% identity with SEQ ID NO: 169. In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 95% identity with SEQ ID NO: 169. In another embodiment, the SerA gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169. Accordingly, In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169. In another embodiment, the genetically engineered bacteria comprise the SerA gene sequence of SEQ ID NO: 169. In yet another embodiment the genetically engineered bacteria comprise a SerA gene sequence which consists of the sequence of SEQ ID NO: 169.

[0520] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding feedback resistant SerA. In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 80% identity with SEQ ID NO: 179. In another embodiment, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 85% identity with SEQ ID NO: 179. In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 90% identity with SEQ ID NO: 179. In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 95% identity with SEQ ID NO: 179. In another embodiment, the SerAfbr gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 179. Accordingly, In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 179. In another embodiment, the genetically engineered bacteria comprise the SerAfbr gene sequence of SEQ ID NO: 179. In yet another embodiment the genetically engineered bacteria comprise a SerAfbr gene sequence which consists of the sequence of SEQ ID NO: 179.

[0521] Table 26 lists exemplary genes encoding kynureninase which are encoded by the genetically engineered bacteria of the disclosure in certain

embodiments.

[0522]

[0523] Table 26. Kynureninase protein sequences

SNPPILLVCSLHASLEIFKQATMKALRKKSVLLTGYLE

YLIKHN YGKD KA AT KKP V VNIITPS H VEERGCQLTITF S VPNKD VFQELE KRG V VCD KRNPNGIRV AP VPLYNS FHDVYKFTNLLTSILDSAETKN*

Shewanella Q8E973 MLLNVKQDFCLAGPGYLLNHSVGRPLKSTEQALKQA SEQ ID NO: FFAPWQES GREPWGQWLGVIDNFT AALAS LFNGQPQ 91 DFCPQVNLSSALTKIVMSLDRLTRDLTRNGGAVVLM

S EIDFPS MGF ALKKALP AS CELRFIPKS LD VTDPN VW D AHICDD VDLVFVS HAYS NTGQQ APL AQIIS L ARERG CLSLVD V AQS AGILPLDLAKLQPDFMIGS S VKWLCS G PGAAYLWVNPAILPECQPQDVGWFSHENPFEFDIHDF RYHPT ALRFWGGTPS I AP Y AI A AHS IE YF ANIGS Q VM REHNLQLMEP V VQ ALDNELVS PQE VD KRS GTIILQFG ERQPQILA AL A A ANIS VDTRS LGIRVS PHI YNDE ADI A RLLGVIKANR*

[0524] * designates the position of the stop codon

[0525] In some embodiments, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 89 through SEQ ID NO: 91. In some embodiments, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 89 through SEQ ID NO: 91. In some embodiments, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 89 through SEQ ID NO: 91. In some embodiments, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 89 through SEQ ID NO: 91. In some embodiments, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 89 through SEQ ID NO: 91. Accordingly, In some embodiments, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 89 through SEQ ID NO: 91. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 89 through SEQ ID NO: 91. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically

engineered bacteria consist of the sequence of one or more of SEQ ID NO: 89 through SEQ ID NO: 91.

[0526] Table 27 lists exemplary codon-optimized kynureninase cassette sequences.

Table 27. Selected codon-optimized kynureninase cassette sequences

Kynureninase Kynureninase protein sequences

protein sequences

kynU(Pseudomona atgacgacccgaaatgattgcctagcgttggatgcacaggacagtctggctccgctgcgccaa s) caatttgcgctgccggagggtgtgatatacctggatggcaattcgctgggcgcacgtccggtag

SEQ ID NO: 92 ctgcgctggctcgcgcgcaggctgtgatcgcagaagaatggggcaacgggttgatccgttcat ggaactctgcgggctggcgtgatctgtctgaacgcctgggtaatcgcctggctaccctgattggt gcgcgcgatggggaagtagttgttactgataccacctcgattaatctgtttaaagtgctgtcagcg gcgctgcgcgtgcaagctacccgtagcccggagcgccgtgttatcgtgactgagacctcgaatt tcccgaccgacctgtatattgcggaagggttggcggatatgctgcaacaaggttacactctgcgt ttggtggattcaccggaagagctgccacaggctatagatcaggacaccgcggtggtgatgctg acgcacgtaaattataaaaccggttatatgcacgacatgcaggctctgaccgcgttgagccacg agtgtggggctctggcgatttgggatctggcgcactctgctggcgctgtgccggtggacctgca ccaagcgggcgcggactatgcgattggctgcacgtacaaatacctgaatggcggcccgggttc gcaagcgtttgtttgggtttcgccgcaactgtgcgacctggtaccgcagccgctgtctggttggtt cggccatagtcgccaattcgcgatggagccgcgctacgaaccttctaacggcattgctcgctat ctgtgcggcactcagcctattactagcttggctatggtggagtgcggcctggatgtgtttgcgca gacggatatggcttcgctgcgccgtaaaagtctggcgctgactgatctgttcatcgagctggttg aacaacgctgcgctgcacacgaactgaccctggttactccacgtgaacacgcgaaacgcggct ctcacgtgtcttttgaacaccccgagggttacgctgttattcaagctctgattgatcgtggcgtgat cggcgattaccgtgagccacgtattatgcgtttcggtttcactcctctgtatactacttttacggaag tttgggatgcagtacaaatcctgggcgaaatcctggatcgtaagacttgggcgcaggctcagttt c&ggtgcgcc&ctctgtt&ctt& aataaaacgaaaggctcagtcgaaagactgggcctttc gttttatctgttg

kynU(Human) atggagccttcatctttagaactgccagcggacacggtgcagcgcatcgcggcggaactgaag SEQ ID NO: 93 tgccatccgactgatgagcgtgtggcgctgcatctggacgaagaagataaactgcgccactttc gtgaatgtttttatattcctaaaattcaagacttgccgccggtagatttgagtctcgttaacaaagat gaaaacgcgatctactttctgggcaactctctgggtctgcaaccaaaaatggttaaaacgtacct ggaggaagaactggataaatgggcaaaaatcgcggcttatggtcacgaagtgggcaagcgtc cttggattactggcgacgagtctattgtgggtttgatgaaagatattgtgggcgcgaatgaaaag gaaattgcactgatgaatgctctgaccgttaatctgcacctgctgatgctgtctttttttaaaccgac cccgaaacgctacaaaatactgctggaagcgaaagcgtttccgtcggatcactatgctatagaa agtcaactgcagttgcatggtctgaatatcgaggaatctatgcgcatgattaaaccgcgtgaggg tgaagaaacgctgcgtattgaagacattctggaagttattgaaaaagaaggtgattctatcgcagt tatactgttttctggcgtgcacttttatacaggtcagcacttcaatatcccggcaatcactaaagcg gggcaggcaaaaggctgctatgttggttttgacctggcgcatgcagtggggaatgttgaactgta tctgcacgattggggcgttgatttcgcgtgttggtgtagctacaaatatctgaacgctggcgcgg gtggcattgctggcgcttttattcacgaaaaacacgcgcacaccattaaaccggctctggttggct ggttcggtcatgagctgagtactcgctttaaaatggataacaaactgcaattgattccgggtgtttg cggcttccgtatcagcaatccgccgattctgctggtttgcagcctgcacgctagtctggaaatcttt aagcaggcgactatgaaagcgctgcgcaaaaaatctgtgctgctgaccggctatctggagtatc tgatcaaacacaattatggcaaagataaagctgcaactaaaaaaccggtagtgaacattatcacc ccctcacacgtggaggagcgcggttgtcagctgactattactttcagtgtacctaataaagatgtg ttccaggaactggaaaaacgcggcgttgtttgtgataaacgtaacccgaatggtattcgcgtggc tcctgtgccgctgtacaattcattccacgatgtttataaattcaccaacctgctgacttctattctcga c&gtgctg&g&ct&&&&&tta aataaaacgaaaggctcagtcgaaagactgggcctttcg ttttatctgttg

kynU(Shewanella) atgctgctgaatgtaaaacaggacttttgcctggcaggcccgggctacctgctgaatcactcggt SEQ ID NO: 94 tggccgtccgctgaaatcaactgagcaagcgctgaaacaagcattttttgctccgtggcaagag agcggtcgtgaaccgtggggccagtggctgggtgttattgataatttcactgctgcgctggcatc tctgtttaatggtcaaccgcaggatttttgtccgcaggttaacctgagcagcgcgctgactaaaatt gtgatgtcactggatcgtctgactcgcgatctgacccgcaatggcggtgctgttgtgctgatgtct gaaatcgatttcccatctatgggcttcgcgttgaaaaaagcgctgccagcgagctgcgaactgc gttttatcccgaaaagtctggacgtgactgatccgaacgtatgggatgcacacatctgtgatgatg tagacctggtttttgtgtctcacgcctatagtaatacgggccaacaggctccgctggcgcaaatca tctctctggcgcgtgaacgtggctgcctgtcactggtggatgtagcgcaatcagcggggattttg ccgctggatctggcgaaactgcaaccggacttcatgatcggcagttcggttaaatggctgtgctc gggccctggtgcggcatatctgtgggttaatccggcgattctgccggaatgtcagccgcaggat gtgggctggttttcacatgagaatccctttgaattcgacatccacgatttccgctaccacccgactg cactgcgcttttggggtggtacgccgtcgatcgcgccttatgcgatcgcggcgcactcgatcga atattttgccaatatcggctcgcaagtgatgcgtgaacacaacctgcaactgatggaaccggtgg ttcaggcgctggacaatgaactggtgagcccgcaggaagtggataaacgctcaggcactattat tctgcaattcggtgaacgtcaaccgcaaattctggcggctctggctgcggcgaacatttcggtgg acactcgttctttggggattcgtgttagtccgcacatttataatgatgaggcggacattgcgcgcct gctgggtgtgatcaaagcaaatcgctaaaaataaaacgaaaggctcagtcgaaagactgggcc tttcgttttatctgttg

The ptet-promoter is in bold, designed Ribosome binding site is underlined, codon- optimized protein coding sequence is in plain text, and the terminator is in italics.

[0527] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 12 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 12 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 12 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 12 or a functional fragment thereof. [0528] In some embodiments, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 92 through SEQ ID NO: 94. In some embodiments, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 92 through SEQ ID NO: 94. In some embodiments, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 92 through SEQ ID NO: 94. In some embodiments, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 92 through SEQ ID NO: 94. In some embodiments, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 92 through SEQ ID NO: 94. Accordingly, In some embodiments, one or more polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 92 through SEQ ID NO: 94. In another embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 92 through SEQ ID NO: 94. In another embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria consists of the sequence of one or more of SEQ ID NO: 92 through SEQ ID NO: 94.

[0529] In some embodiments, the genetically engineered bacteria comprise gene sequences for the expression of one or more modified kynureninases, e.g., human kynureninases modified by substitutions, e.g., in the substrate recognition site or any location that may affect substrate specificity, which may include kynurenine degrading activity or 3'-hydroxy-kynurenine degrading activity, as described in US2016/0058845, the contents of which is herein incorporated by reference in its entirety.

[0530] The genetically engineered bacteria may comprise any suitable gene for producing kynureninase. In some embodiments, the gene for producing kynureninase is modified and/or mutated, e.g., to enhance stability, increase kynureninase production. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above.

[0531] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0532] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan is modified and/or mutated, e.g., to enhance stability, or increase

tryptophan/tryptophan metabolite production or catalysis. [0533] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the degradation of tryptophan are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0534] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the production of a short chain fatty acid.

[0535] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0536] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the degradation of tryptophan further comprise one or more gene sequences for the catabolism of adenosine.

[0537] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0538] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0539] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Producing Kynurenic Acid

[0540] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible

transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine- oxoglutarate transaminase.

[0541] In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, e.g., the gut or the tumor microenvironment, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, inflammation or an inflammatory response, immune suppression, e.g., in cancer, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[0542] In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYNA ratio, e.g. in the circulation. In some embodiments, the TRP:KYNA ratio is increased. In some embodiments, TRP:KYNA ratio is decreased. In some embodiments, the genetically engineered bacteria the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the KYNA:QUIN ratio..

[0543] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenic acid, which are bacterially derived. In some embodiments, the enzymes for producing kynureic acid are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some

embodiments, the enzymes are derived from the species listed in table S7 of Vujkovic- Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

[0544] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters, gene sequence(s) encoding kynureninase, and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding one or more kynurenine

aminotransferases.

[0545] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenic acid from tryptophan. Non- limiting example of such gene sequence(s) are shown FIG. 16F and described elsewhere herein. In some

embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ID01(indoleamine 2,3-dioxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDOl from homo sapiens. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 (tryptophan 2,3- dioxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TD02 from homo sapiens. In some

embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In some

embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of idol and/or tdo2 and/or bna2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with IDOL In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with TD02. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In some

embodiments, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cclb2 and/or aadat and/or got2.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine— oxoglutarate transaminase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cclb2 and/or aadat and/or got2.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.

[0546] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from homo sapiens. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from homo sapiens. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB 1 (Kynurenine— oxoglutarate transaminase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB 1 from homo sapiens). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine— oxoglutarate transaminase 3) In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from homo sapiens. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cclbl and/or cclb2 and/or aadat and/or got2.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of . cclbl and/or cclb2 and/or aadat and/or got2.

[0547] In any of these embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B. and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0548] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenic acid is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0549] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenic acid is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenic acid is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenic acid is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0550] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenic acid may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of kynurenic acid are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome. [0551] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the production of a short chain fatty acid.

[0552] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0553] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion or display of an anti-PD 1 antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the secretion or display of an anti-PD- Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of kynurenic acid further comprise one or more gene sequences for the catabolism of adenosine.

[0554] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0555] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0556] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Producing Indole Tryptophan Metabolites and Tryptamine

[0557] In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of indole metabolites and/or tryptamine. Exemplary circuits for the production of indole metabolites/derivatives are shown in FIG. 41A through FIG. 41H, FIG. 42A through FIG. 42E, and FIG. 43A though FIG 43B, and FIG. 45A through FIG. 45E.

[0558] In in any of these embodiments, the expression of the gene sequences for the production of the indole and other tryptophan metabolites, including, but not limited to, tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, , indole, indole acetic acid FICZ, indole-3-propionic acid is under the control of an inducible promoter. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0559] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites is operably linked to a RBS, enhancer or other regulatory sequence. In some

embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0560] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0561] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the production of a short chain fatty acid.

[0562] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the production of a butyrate. In any of the

embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of an antiinflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0563] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan and indole metabolites further comprise one or more gene sequences for the catabolism of adenosine.

[0564] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0565] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0566] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Tryptamine

[0567] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is derived from the direct decarboxylation of tryptophan. Tryptophan is converted to indole-3- acetic acid (IAA) via the enzymes tryptophan monooxygenase (laaM) and indole-3- acetamide hydrolase (IaaH), which constitute the indole- 3 -acetamide (IAM) pathway, see eg., FIG. 17D, FIG. 6A and FIG. 6B.

[0568] A non-limiting example of such as strain is shown in FIG. 18B. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s). e.g., from

Catharanthus roseus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s). e.g., from Catharanthus roseus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s). e.g., from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from

Ruminococcus Gnavus. [0569] Another non-limiting example of such as strain is shown in FIG. 21A. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc(tryptophan decarboxylase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Clostridium sporogenes.

[0570] Table 28 and 29 lists exemplary sequences for tryptamine production in genetically engineered bacteria.

[0571] In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites..

[0572] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0573] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0574] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0575] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the production of a short chain fatty acid.

[0576] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the catabolism of branched chain amino acids. [0577] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the catabolism of adenosine.

[0578] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. [0579] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0580] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Indole-3-acetaldehyde and FICZ

[0581] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole- 3 -acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from

tryptophan are shown in FIG. 16B.

[0582] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In some embodiments, the (L-tryptophan aminotransferase is from S. cerevisiae. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from

Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and ipdC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and ipdC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and ipdC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and ipdC.

[0583] Further exemplary gene cassettes for the production of produce indole-3- acetaldehyde and FICZ from tryptophan are shown in FIG. 16C. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (e.g., from Clostridium sporogenes) and tynA.

[0584] In any of these embodiments, the genetically engineered bacteria which produce produce indole- 3 -acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole- 3 -acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0585] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. [0586] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0587] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0588] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the production of a short chain fatty acid.

[0589] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0590] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the catabolism of adenosine.

[0591] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0592] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0593] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Indole-3 -acetic acid

[0594] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole Acetic Acid, e.g., via a tryptophan aminotransferase cassette. A non-limiting example of such a tryptophan aminotransferase expressed by the genetically engineered bacteria is in Table 15. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter, and further produce Indole- 3 -aldehyde and Indole Acetic Acid from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan and/or indole metabolite exporter.

[0595] The genetically engineered bacteria may comprise any suitable gene for producing Indole- 3 -aldehyde and/or Indole Acetic Acidand/or Tryptamine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase Indole- 3 -aldehyde and/or Indole Acetic Acidand/or

Tryptamine production, and/or increase ant i- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite , e.g., comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole- 3 -aldehyde and/or Indole Acetic Acidand/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.

[0596] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole- 3 -acetic acid. Non-limiting example of such gene sequence(s) are shown in FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, and FIG. 17E.

[0597] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from S. cerevisae). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP- A0274). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole- 3 -acetaldehyde dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl (Indole- 3 -acetaldehyde oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole- 3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or staO and/or trpDH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from iadl and/or aaol.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole- 3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or staO and in combination with one or more sequences encoding enzymes selected from iadl and/or aaol (see, e.g., FIG. 17A).

[0598] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 17B. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole- 3 -acetaldehyde dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl (Indole- 3 -acetaldehyde oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAOl from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iadl and/or aaol. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iadl and/or aaol. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iadl and/or aaol.

[0599] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 17D. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES- 2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and/or ipdC and/or iadl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and ipdC and iadl. [0600] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 17C. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole-3-pyruvate monooxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from Enterobacter cloacae. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In some embodiments, the (L-tryptophan aminotransferase is from S. cerevisiae. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and yuc2.In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In some

embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and yuc2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and yuc2.. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or staO or trpDH and yuc2.

[0601] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 17D. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2- monooxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH.

[0602] Another non- limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 17E. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3

(indoleacetaldoxime dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 from

Arabidopis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nitl (Nitrilase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nitl from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71al3. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from

Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nitl and/or iaaH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3 and nitl and/or iaaH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3, and nitl and/or iaaH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3 and nitl and iaaH. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3 and nitl and iaaH.

[0603] In any of these embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0604] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0605] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid is operably linked to a constitutive promoter. In some embodiments, the

constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the

embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0606] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome. [0607] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the production of a short chain fatty acid.

[0608] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0609] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the secretion or display of an anti-PD- Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole- 3 -acetic acid further comprise one or more gene sequences for the catabolism of adenosine.

[0610] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0611] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0612] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Indole-3-acetonitrile

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetonitrile from tryptophan. A non-limiting example of such gene sequence(s) which allow in which the genetically engineered bacteria to produce indole-3- acetonitrile from tryptophan is depicted in FIG. 16D.

[0613] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 (indoleacetaldoxime dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71al3 from Arabidopis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71al3.

[0614] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase) In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71al3. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71al3.

[0615] In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0616] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- acetonitrile is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0617] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- acetonitrile is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the

embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0618] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- acetonitrile may be codon optimized, e.g., to improve expression in the host

microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- acetonitrile are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0619] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the production of a short chain fatty acid.

[0620] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0621] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the secretion or display of an anti-PD- LI antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-acetonitrile further comprise one or more gene sequences for the catabolism of adenosine.

[0622] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0623] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0624] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety. lndole-3 -propionic acid (IP A)

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-propionic acid from tryptophan. FIG. 18A and FIG 18B depict schematics of exemplary circuits for the production of indole-3-propionic acid.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase from Rubrivivax benzoatilyticus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole- 3 -aery late reductase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole- 3 -aery late reductase from Clostridum botulinum. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole- 3 -aery late reductase. In some embodiments, the indole-3-propionate-producing strain optionally produces tryptophan from a chorismate precursor, and the strain optionally comprises additional circuits for tryptophan production and/or tryptophan uptake/transport s described herein. FIG. 21C depicts another non-limiting example of an indole-3-propionate-producing strain. The genetically engineered bacteria comprise a circuit, comprising trpDH

(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indol-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3- lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole- 3 -lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3- lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or Acul: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3- acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldHl and/or fldH2 (indole- 3 -lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole- 3 -lactate) (see, e.g., FIG. 6A and FIG. 6B).

In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA (indole-3-propionyl- CoA:indole-3-lactate CoA transferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA from

Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC (indole-3-lactate dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD (indole-3-acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD from

Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding Acul (acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding Acul from Rhodobacter sphaeroides. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldHl (3-lactate dehydrogenase 1). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldHl from Clostridium sporogenes,. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 (indole- 3 -lactate dehydrogenase 2). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 from Clostridium sporogenes). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acul and/or fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acul and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acul and fldHl. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acul and fldH2.

[0625] In any of these embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0626] In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 different tryptophan metabolites. In certain embodiments, the bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan

metabolites selected from tryptamine and/or indole-3 acetaladehyde, indole- 3acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3- propionic acid.

[0627] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- propionic acid is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0628] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- propionic acid is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0629] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- propionic acid may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole-3- propionic acid are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0630] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the production of a short chain fatty acid.

[0631] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0632] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti- TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3- propionic acid further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole-3-propionic acid further comprise one or more gene sequences for the catabolism of adenosine.

[0633] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0634] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0635] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Indole

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 16G and described elsewhere herein. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA

(tryptophanase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA from E. coli.

[0636] In any of these embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0637] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0638] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole is modified and/or mutated, e.g., to enhance stability, or increase

tryptophan/tryptophan metabolite production or catalysis.

[0639] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the

microorganism chromosome.

[0640] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the production of a short chain fatty acid.

[0641] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0642] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole further comprise one or more gene sequences for the catabolism of adenosine.

[0643] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0644] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0645] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Other indole metabolites

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. Non-limiting example of such gene sequence(s) are shown FIG. 16G and described elsewhere herein. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 (myrosinase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2from Arabidopsis thaliana.

[0646] In any of these embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 1 (A-D), FIG. 19A and/or FIG. 19B and described elsewhere herein. In some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.

[0647] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole metabolites is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0648] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole metabolites is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole metabolites is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole metabolites is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis. [0649] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole metabolites may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indole metabolites are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

[0650] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the production of a short chain fatty acid. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0651] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the secretion or display of an anti-PD- Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indole metabolites further comprise one or more gene sequences for the catabolism of adenosine.

[0652] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0653] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0654] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

Tryptophan Catabolic Pathway Enzymes

[0655] Table 28 and 29 comprises polypeptide sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure.

Table 28 Tryptophan Pathway Enzymes

ATCTGACCTTGGTTTTCAACGCCTGGCTGAAGCTATTAAGG ATAAACGCAAGGCTTTAGGGCTGGCT

Tryptophan ATGAGTCAAGTGATTAAGAAGAAACGTAACACCTTTATGA Decarboxylase (EC TCGGAACGGAGTACATTCTTAACAGTACACAATTGGAGGA 4.1.1.28) Chain A, AGCGATTAAATCATTCGTACATGATTTCTGCGCAGAGAAGC Ruminococcus ATGAGATCCATG ATCAACCTGTGGTAGTAGAAGCTAAAGA Gnavus Tryptophan

ACATCAGGAGGACAAAATCAAACAAATCAAAATCCCGGAA

Decarboxylase Rum

AAGGGACGTCCTGTAAATGAAGTCGTTTCTGAGATGATGA

gna_01526 (alpha- fmt); codon ATG AAGTGTATCGCTACCGCGGAGACGCCAACCATCCTCG optimized for the CTTTRNTCM GTGCCCGGACCTGCAAGCAGTGTGTCGTG expression in E. coli GTTGGGGGATATTATGACGTCCGCCTACAATATTCATGCTG

GAGGOX: AAAGCTGGCAiXGAl u

SEQ ID NO: 96 GGAAGTTCTGAAGTGGTTACICAAAGCAAGTGGGGTTCACA

GAAAATCCAGGTGGCGTATTTGTGTCGGGCGGTTCAATGG

CGAATATTACGCJ( ACTTACTGCGCK:TCGTGACAATAAACTG

ACCGACATTAACCTTCATTTGGGAACTGCTTATATTAGTGA

CC AG ACTC AT AGTTC AGTTGCG A A AGG ATT ACGC ATT ATTG

GAATCACTGACAGTCGCATCCGTCGCATTCCCACTAACTCC

CACTTCCAGATGGATACCACCAAGCTGGAGGAAGCCATCG

AGACCGACAAGAAGTCTGC : TACATTCCGTTCGTCGTTATC

GGAACACJCAGGTACCACCAACACTGGTTCGATTGACCCCC TGACAGAAATCTCTGCGTTATGTAAGAAGCATGACATGTG

ί:ΓΓΊ^,Ίx:;AΊ^{^}AΊ ; :ϊAί: :χ}A( :XϊΊ^{^}AΊx:χ}AC}c;Ί^,A(JΊx:ΓΓΊ :ΊXϊ(Jrί: ]^,

CACCTAAGTACAAGAGCCTTCTTACCGGAACCGGCTTGGCT GACAGTATTTCGTGGGATGCTCATAAATGGTTGTTCCAAAC GTAOGGC^'l GTGCAA iGGTAi TTGTCAAAGA I Υί X ^'G I A A i f TATTCCACTCTTTTCATGTGAATCCCGAGTATCTTAAGGAT

c;ix:x}AAAAC LAc:;Ai L i AA(: :ii^,i^ Ai Ac Aix:x}GAi Aixx}

GCATGGAGCTGACGCGCX:CTGCACGCGGTCTTAAATTGTG

GCTTACJ "i ^' AC AGGTCCTTGGATCTGACTTGATTGGG AGTG

( ^'A Γ^'!Χ^ Α(\ Γ( Κ = Γ^'Γ^' { A C :C rGC =CA( ^r r c xH^x

AGCATTGAATCCAAAGAAAGACTGGGAGATCGTTTCTCCA

GCTCAGATGGCTATGATTAATTTCCGTTATGCCCCTAAGGA

TTTAACCAAAGAGGAACAGGATATTCrcAATGAAAAGATC

TCCCACCGCATTTTAGAGAGCGGATACGCTGCAATTTTCAC

Ί AC TGTATTA C GGG\AGACCG I 1 I 1 ACGC^'ATCTGTGC AA

TTCACCCGGAGGCAACTCAAGAGGATATGCAACACACAAT

CG ACTTATTAG ACC A AT ACGGTCGTG AA ATCT AT ACCG AG

ATGAAG AAAGCG

Tdc (tdc from C. ATGGGTTCTATTGACTCGACGAATGTGGCCATGTCTAATTC roseus) TCCTGTTGGCGAGTTTAAGCCCCTTGAAGCAGAAGAGTTCC

GTAAACAGGCACACCGCATGGTGGATTTTATTGCGGATTAT

SEQ ID NO: 171 TACAAGAACGTAGAAACATACCCGGTCCTTTCCGAGGTTG

AACCCGGCTATCTGCGCAAACGTATTCCCGAAACCGCACC ATACCTGCCGGAGCCACTTGATGATATTATGAAGGATATTC

AAAAGGACATTATCCCCGGAATGACGAACTGGATGTCCCC

GAACTTTTACGCCTTCTTCCCGGCCACAGTTAGCTCAGCAG

CTTTCTTGGGGGAAATGCTTTCAACGGCCCTTAACAGCGTA

GGATTTACCTGGGTCAGTTCCCCGGCAGCGACTGAATTAGA

GATGATCGTTATGGATTGGCTTGCGCAAATTTTGAAACTTC

CAAAAAGCTTTATGTTCTCCGGAACCGGGGGTGGTGTCATC

CAAAACACTACGTCAGAGTCGATCTTGTGCACTATTATCGC

GGCCCGTGAACGCGCCTTGGAAAAATTGGGCCCTGATTCA

ATTGGTAAGCTTGTCTGCTATGGGTCCGATCAAACGCACAC

AATGTTTCCGAAAACCTGTAAGTTAGCAGGAATTTATCCGA

ATAATATCCGCCTTATCCCTACCACGGTAGAAACCGACTTT

GGCATCTCACCGCAGGTACTTCGCAAGATGGTCGAAGACG

ACGTCGCTGCGGGGTACGTTCCCTTATTTTTGTGTGCCACC

TTGGGAACGACATCAACTACGGCAACAGATCCTGTAGATT

CGCTGTCCGAAATCGCAAACGAGTTTGGTATCTGGATTCAT

GTCGACGCCGCATATGCTGGATCGGCTTGCATCTGCCCAGA

ATTTCGTCACTACCTTGATGGCATCGAACGTGTGGATTCCT

TATCGCTGTCTCCCCACAAATGGCTTTTAGCATATCTGGAT

TGCACGTGCTTGTGGGTAAAACAACCTCACCTGCTGCTTCG

CGCTTTAACGACTAATCCCGAATACTTGAAGAATAAACAG

AGTGATTTAGATAAGGTCGTGGATTTTAAGAACTGGCAGA

TCGCAACAGGACGTAAGTTCCGCTCTTTAAAACTTTGGTTA

ATTCTGCGTTCCTACGGGGTAGTTAACCTGCAAAGTCATAT

CCGTAGTGATGTAGCGATGGGGAAGATGTTTGAGGAATGG

GTCCGTTCCGATAGCCGCTTTGAAATCGTCGTGCCACGTAA

TTTTTCGCTTGTATGCTTTCGCTTGAAACCGGATGTATCTAG

TTTACATGTCGAGGAGGTCAACAAGAAGTTGTTGGATATG

CTTAACTCCACCGGTCGCGTATATATGACGCATACAATTGT

TGGCGGAATCTATATGTTACGTTTGGCTGTAGGTAGCAGCT

TGACAGAGGAACATCACGTGCGCCGCGTTTGGGACTTGAT

CCAGAAGCTTACGGACGACCTGCTTAAAGAGGCGTGA

Tdc (tdc from ATGAAATTTTGGCGCAAGTATACGCAACAGGAGATGGATG

Clostridium AGAAAATCACAGAATCGCTTGAGAAGACATTAAATTACGA sporogenes) TAACACGAAAACCATCGGCATCCCAGGTACTAAGCTGGAT

GATACTGTATTTTATGACGATCACTCCTTCGTTAAGCACTC

SEQ ID NO: 173 TCCCTATTTACGTACGTTCATCCAAAACCCTAATCACATTG

GTTGTCACACGTACGATAAAGCAGACATCTTGTTTGGCGGC

ACGTTTGACATCGAACGCGAACTGATTCAGCTTTTGGCCAT

CGATGTCTTAAACGGAAATGATGAGGAATTCGATGGATAT

GTGACACAGGGGGGAACCGAGGCGAATATTCAGGCAATGT

GGGTTTATCGTAACTATTTCAAAAAAGAACGTAAAGCAAA

ACATGAGGAAATCGCAATCATCACGAGCGCGGATACCCAT

TACAGTGCATATAAGGGGAGCGACTTGCTGAACATTGATA

TTATCAAGGTCCCAGTAGACTTCTATTCGCGTAAGATCCAG

GAGAACACGTTAGACTCGATTGTCAAGGAGGCGAAGGAAA

TTGGAAAGAAGTACTTCATTGTCATCTCAAACATGGGTACG

ACTATGTTTGGCAGTGTAGACGACCCTGATCTTTATGCTAA

CATTTTTGATAAGTATAACTTAGAATACAAAATCCACGTCG ATGGAGCTTTTGGGGGTTTCATTTATCCTATCGATAATAAG

GAGTGCAAAACAGATTTCTCGAACAAGAACGTCTCATCCA

TCACGCTTGACGGTCACAAAATGCTTCAAGCCCCCTATGGG

ACTGGTATCTTCGTGTCACGTAAGAACTTGATCCATAACAC

CCTGACAAAGGAAGCAACGTATATTGAAAACCTGGACGTT

ACCCTGAGTGGGTCCCGCTCCGGATCCAACGCCGTTGCGAT

CTGGATGGTTTTAGCCTCTTATGGCCCCTACGGGTGGATGG

AGAAGATTAACAAGTTGCGCAATCGCACTAAGTGGCTTTG

CAAGCAGCTTAACGACATGCGCATCAAATACTATAAGGAG

GATAGCATGAATATCGTCACGATTGAAGAGCAATACGTAA

ATAAAGAGATTGCAGAGAAATACTTCCTTGTGCCTGAAGT

ACACAATCCTACCAACAATTGGTACAAGATTGTAGTCATG

GAACATGTTGAACTTGACATCTTGAACTCCCTTGTTTATGA

TTTACGTAAATTCAACAAGGAGCACCTGAAGGCAATGTGA

trpDH ATGCTGTTATTCGAGACTGTGCGTGAAATGGGTCATGAGCA

SEQ ID NO: 175 AGTCCTTTTCTGTCATAGCAAGAATCCCGAGATCAAGGCAA

TTATCGCAATCCACGATACCACCTTAGGACCGGCTATGGGC

GCAACTCGTATCTTACCTTATATTAATGAGGAGGCTGCCCT

GAAAGATGCATTACGTCTGTCCCGCGGAATGACTTACAAA

GCAGCCTGCGCCAATATTCCCGCCGGGGGCGGCAAAGCCG

TCATCATCGCTAACCCCGAAAACAAGACCGATGACCTGTT

ACGCGCATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTT

TCATCACCGGGCAGGACGTTAACATTACGCCCGACGACGT

TCGCACTATTTCGCAGGAGACTAAGTACGTGGTAGGCGTCT

CAGAAAAGTCGGGAGGGCCGGCACCTATCACCTCTCTGGG

AGTATTTTTAGGCATCAAAGCCGCTGTAGAGTCGCGTTGGC

AGTCTAAACGCCTGGATGGCATGAAAGTGGCGGTGCAAGG

ACTTGGGAACGTAGGAAAAAATCTTTGTCGCCATCTGCATG

AACACGATGTACAACTTTTTGTGTCTGATGTCGATCCAATC

AAGGCCGAGGAAGTAAAACGCTTATTCGGGGCGACTGTTG

TCGAACCGACTGAAATCTATTCTTTAGATGTTGATATTTTT

GCACCGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT

CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCGAAT

AACCAGCTGGAGAACGAGCAACTTCATTCGCAGATGCTTG

CGAAAAAGGGTATTCTTTACTCACCAGACTACGTTATCAAT

GCAGGAGGACTTATCAATGTTTATAACGAAATGATCGGAT

ATGACGAGGAAAAAGCATTCAAACAAGTTCATAACATCTA

CGATACGTTATTAGCGATTTTCGAAATTGCAAAAGAACAA

GGTGTAACCACCAACGACGCGGCCCGTCGTTTAGCAGAGG

ATCGTATCAACAACTCCAAACGCTCAAAGAGTAAAGCGAT

TGCGGCGTGA

ipdC ATGCGTACACCCTACTGTGTCGCCGATTATCTTTTAGATCG

TCTGACGGACTGCGGGGCCGATCACCTGTTTGGCGTACCGG

SEQ ID NO: 176 GCGATTACAACTTGCAGTTTCTGGACCACGTCATTGACTCA

CCAGATATCTGCTGGGTAGGGTGTGCGAACGAGCTTAACG

CGAGCTACGCTGCTGACGGATATGCGCGTTGTAAAGGCTTT

GCTGCACTTCTTACTACCTTCGGGGTCGGTGAGTTATCGGC

GATGAACGGTATCGCAGGCTCGTACGCTGAGCACGTCCCG

GTATTACACATTGTGGGAGCTCCGGGTACCGCAGCTCAAC AGCGCGGAGAACTGTTACACCACACGCTGGGCGACGGAGA

ATTCCGCCACTTTTACCATATGTCCGAGCCAATTACTGTAG

CCCAGGCTGTACTTACAGAGCAAAATGCCTGTTACGAGAT

CGACCGTGTTTTGACCACGATGCTTCGCGAGCGCCGTCCCG

GGTATTTGATGCTGCCAGCCGATGTTGCCAAAAAAGCTGC

GACGCCCCCAGTGAATGCCCTGACGCATAAACAAGCTCAT

GCCGATTCCGCCTGTTTAAAGGCTTTTCGCGATGCAGCTGA

AAATAAATTAGCCATGTCGAAACGCACCGCCTTGTTGGCG

GACTTTCTGGTCCTGCGCCATGGCCTTAAACACGCCCTTCA

GAAATGGGTCAAAGAAGTCCCGATGGCCCACGCTACGATG

CTTATGGGTAAGGGGATTTTTGATGAACGTCAAGCGGGATT

TTATGGAACTTATTCCGGTTCGGCGAGTACGGGGGCGGTA

AAGGAAGCGATTGAGGGAGCCGACACAGTTCTTTGCGTGG

GGACACGTTTCACCGATACACTGACCGCTGGATTCACACAC

CAACTTACTCCGGCACAAACGATTGAGGTGCAACCCCATG

CGGCTCGCGTGGGGGATGTATGGTTTACGGGCATTCCAATG

AATCAAGCCATTGAGACTCTTGTCGAGCTGTGCAAACAGC

ACGTCCACGCAGGACTGATGAGTTCGAGCTCTGGGGCGAT

TCCTTTTCCACAACCAGATGGTAGTTTAACTCAAGAAAACT

TCTGGCGCACATTGCAAACCTTTATCCGCCCAGGTGATATC

ATCTTAGCAGACCAGGGTACTTCAGCCTTTGGAGCAATTGA

CCTGCGCTTACCAGCAGACGTGAACTTTATTGTGCAGCCGC

TGTGGGGGTCTATTGGTTATACTTTAGCTGCGGCCTTCGGA

GCGCAGACAGCGTGTCCAAACCGTCGTGTGATCGTATTGA

CAGGAGATGGAGCAGCGCAGTTGACCATTCAGGAGTTAGG

CTCGATGTTACGCGATAAGCAGCACCCCATTATCCTGGTCC

TGAACAATGAGGGGTATACAGTTGAACGCGCCATTCATGG

TGCGGAACAACGCTACAATGACATCGCTTTATGGAATTGG

ACGCACATCCCCCAAGCCTTATCGTTAGATCCCCAATCGGA

ATGTTGGCGTGTGTCTGAAGCAGAGCAACTGGCTGATGTTC

TGGAAAAAGTTGCTCATCATGAACGCCTGTCGTTGATCGAG

GTAATGTTGCCCAAGGCCGATATCCCTCCGTTACTGGGAGC

CTTGACCAAGGCTTTAGAAGCCTGCAACAACGCTTAA

ladl ATGCCCACCTTGAACTTGGACTTACCCAACGGTATTAAGAG

CACGATTCAGGCAGACCTTTTCATCAATAATAAGTTTGTGC

SEQ ID NO: 177 CGGCGCTTGATGGGAAAACGTTCGCAACTATTAATCCGTCT

ACGGGGAAAGAGATCGGACAGGTGGCAGAGGCTTCGGCG

AAGGATGTGGATCTTGCAGTTAAGGCCGCGCGTGAGGCGT

TTGAAACTACTTGGGGGGAAAACACGCCAGGTGATGCTCG

TGGCCGTTTACTGATTAAGCTTGCTGAGTTGGTGGAAGCGA

ATATTGATGAGTTAGCGGCAATTGAATCACTGGACAATGG

GAAAGCGTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTG

TGGCCGCAAACTTACGTTACTACGGCGGTTGGGCTGATAA

AAACCACGGTAAAGTCATGGAGGTAGACACAAAGCGCCTG

AACTATACCCGCCACGAGCCGATCGGGGTTTGCGGACAAA

TCATTCCGTGGAATTTCCCGCTTTTGATGTTTGCATGGAAG

CTGGGTCCCGCTTTAGCCACAGGGAACACAATTGTGTTAAA

GACTGCCGAGCAGACTCCCTTAAGTGCTATCAAGATGTGTG

AATTAATCGTAGAAGCCGGCTTTCCGCCCGGAGTAGTTAAT GTGATCTCGGGATTCGGACCGGTGGCGGGGGCCGCGATCT

CGCAACACATGGACATCGATAAGATTGCCTTTACAGGATC

GACATTGGTTGGCCGCAACATTATGAAGGCAGCTGCGTCG

ACTAACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAAAT

CCCCGAATATCATTTTCAAAGATGCCGACCTTGACCAAGCT

GTTCGCTGGAGCGCCTTCGGTATCATGTTTAACCACGGACA

ATGCTGCTGCGCTGGATCGCGCGTATATGTGGAAGAATCC

ATCTATGACGCCTTCATGGAAAAAATGACTGCGCATTGTAA

GGCGCTTCAAGTTGGAGATCCTTTCAGCGCGAACACCTTCC

AAGGACCACAAGTCTCGCAGTTACAATACGACCGTATCAT

GGAATACATCGAATCAGGGAAAAAAGATGCAAATCTTGCT

TTAGGCGGCGTTCGCAAAGGGAATGAGGGGTATTTCATTG

AGCCAACTATTTTTACAGACGTGCCGCACGACGCGAAGAT

TGCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGTCGA

AATTTAAGGACGAAAAAGATCTGATCCGTATCGCAAATGA

TTCTATTTATGGTTTAGCTGCGGCAGTCTTTTCCCGCGACAT

CAGCCGCGCGATCGAGACAGCACACAAACTGAAAGCAGGC

ACGGTCTGGGTCAACTGCTATAATCAGCTTATTCCGCAGGT

GCCATTCGGAGGGTATAAGGCTTCCGGTATCGGCCGTGAG

TTGGGGGAATATGCCTTGTCTAATTACACAAATATCAAGGC

CGTCCACGTTAACCTTTCTCAACCGGCGCCCATTTGA

fldA ATGGAAAACAACACCAATATGTTCTCTGGAGTGAAGGTGA

TCGAACTGGCCAACTTTATCGCTGCTCCGGCGGCAGGTCGC

TTCTTTGCTGATGGGGGAGCAGAAGTAATTAAGATCGAAT

SEQ ID NO: 187 CTCCAGCAGGCGACCCGCTGCGCTACACGGCCCCATCAGA

AGGACGCCCGCTTTCTCAAGAGGAAAACACAACGTATGAT

TTGGAAAACGCGAATAAGAAAGCAATTGTTCTGAACTTAA

AATCGGAAAAAGGAAAGAAAATTCTTCACGAGATGCTTGC

TGAGGCAGACATCTTGTTAACAAATTGGCGCACGAAAGCG

TTAGTCAAACAGGGGTTAGATTACGAAACACTGAAAGAGA

AGTATCCAAAATTGGTATTTGCACAGATTACAGGATACGG

GGAGAAAGGACCCGACAAAGACCTGCCTGGTTTCGACTAC

ACGGCGTTTTTCGCCCGCGGAGGAGTCTCCGGTACATTATA

TGAAAAAGGAACTGTCCCTCCTAATGTGGTACCGGGTCTG

GGTGACCACCAGGCAGGAATGTTCTTAGCTGCCGGTATGG

CTGGTGCGTTGTATAAGGCCAAAACCACCGGACAAGGCGA

CAAAGTCACCGTTAGTCTGATGCATAGCGCAATGTACGGC

CTGGGAATCATGATTCAGGCAGCCCAGTACAAGGACCATG

GGCTGGTGTACCCGATCAACCGTAATGAAACGCCTAATCCT

TTCATCGTTTCATACAAGTCCAAAGATGATTACTTTGTCCA

AGTTTGCATGCCTCCCTATGATGTGTTTTATGATCGCTTTAT

GACGGCCTTAGGACGTGAAGACTTGGTAGGTGACGAACGC

TACAATAAGATCGAGAACTTGAAGGATGGTCGCGCAAAAG

AAGTCTATTCCATCATCGAACAACAAATGGTAACGAAGAC

GAAGGACGAATGGGACAAGATTTTTCGTGATGCAGACATT

CCATTCGCTATTGCCCAAACGTGGGAAGATCTTTTAGAAGA

CGAGCAGGCATGGGCCAACGACTACCTGTATAAAATGAAG

TATCCCACAGGCAACGAACGTGCCCTGGTACGTTTACCTGT

GTTCTTCAAAGAAGCTGGACTTCCTGAATACAACCAGTCGC CACAGATTGCTGAGAATACCGTGGAAGTGTTAAAGGAGAT

GGGATATACCGAGCAAGAAATTGAGGAGCTTGAGAAAGAC

AAAGACATCATGGTACGTAAAGAGAAATGA

fldB ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAAGGCT

AAACACTATCTGCGCGAGATCACAGCTAAACACTACAAGG

SEQ ID NO: 188 AAGCGTTAGAGGCTAAAGAGCGTGGGGAGAAAGTGGGTTG

GTGTGCCTCTAACTTCCCCCAAGAGATTGCAACCACGTTGG

GTGTAAAGGTTGTTTATCCCGAAAACCACGCCGCCGCCGTA

GCGGCACGTGGCAATGGGCAAAATATGTGCGAACACGCGG

AGGCTATGGGATTCAGTAATGATGTGTGTGGATATGCACGT

GTAAATTTAGCCGTAATGGACATCGGCCATAGTGAAGATC

AACCTATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAAT

ATCTGCAATCAGATGATTAAATGGTATGAACACATTGCAA

AAACGTTGGATATTCCTATGATCCTTATCGATATTCCATAT

AATACTGAGAACACGGTGTCTCAGGACCGCATTAAGTACA

TCCGCGCCCAGTTCGATGACGCTATCAAGCAACTGGAAGA

AATCACTGGCAAAAAGTGGGACGAGAATAAATTCGAAGAA

GTGATGAAGATTTCGCAAGAATCGGCCAAGCAATGGTTAC

GCGCCGCGAGCTACGCGAAATACAAACCATCACCGTTTTC

GGGCTTTGACCTTTTTAATCACATGGCTGTAGCCGTTTGTG

CTCGCGGCACCCAGGAAGCCGCCGATGCATTCAAAATGTT

AGCAGATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT

ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCGAGG

GCATCGCTTGTTGGCCTTATCTGCGCCACAAGTTGACGAAA

CTGAGTGAATATGGAATGAACGTCACAGCTACGGTGTACG

CCGAAGCTTTTGGGGTTATTTACGAAAACATGGATGAACTG

ATGGCCGCTTACAATAAAGTGCCTAACTCAATCTCCTTCGA

GAACGCGCTGAAGATGCGTCTTAATGCCGTTACAAGCACC

AATACAGAAGGGGCTGTTATCCACATTAATCGCAGTTGTA

AGCTGTGGTCAGGATTCTTATACGAACTGGCCCGTCGTTTG

GAAAAGGAGACGGGGATCCCTGTTGTTTCGTTCGACGGAG

ATCAAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGAC

ACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCGAAAA

AAGAAGCAGAGTGA

fldC ATGTCGAATAGTGACAAGTTTTTTAACGACTTCAAGGACAT

TGTGGAAAACCCAAAGAAGTATATCATGAAGCATATGGAA

SEQ ID NO: 189 CAAACGGGACAAAAAGCCATCGGTTGCATGCCTTTATACA

CCCCAGAAGAGCTTGTCTTAGCGGCGGGTATGTTTCCTGTT

GGAGTATGGGGCTCGAATACTGAGTTGTCAAAAGCCAAGA

CCTACTTTCCGGCTTTTATCTGTTCTATCTTGCAAACTACTT

TAGAAAACGCATTGAATGGGGAGTATGACATGCTGTCTGG

TATGATGATCACAAACTATTGCGATTCGCTGAAATGTATGG

GACAAAACTTCAAACTTACAGTGGAAAATATCGAATTCAT

CCCGGTTACGGTTCCACAAAACCGCAAGATGGAGGCGGGT

AAAGAATTTCTGAAATCCCAGTATAAAATGAATATCGAAC

AACTGGAAAAAATCTCAGGGAATAAGATCACTGACGAGAG

CTTGGAGAAGGCTATTGAAATTTACGATGAGCACCGTAAA

GTCATGAACGATTTCTCTATGCTTGCGTCCAAGTACCCTGG

TATCATTACGCCAACGAAACGTAACTACGTGATGAAGTCA GCGTATTATATGGACAAGAAAGAACATACAGAGAAGGTAC

GTCAGTTGATGGATGAAATCAAGGCCATTGAGCCTAAACC

ATTCGAAGGAAAACGCGTGATTACCACTGGGATCATTGCA

GATTCGGAGGACCTTTTGAAAATCTTGGAGGAGAATAACA

TTGCTATCGTGGGAGATGATATTGCACACGAGTCTCGCCAA

TACCGCACTTTGACCCCGGAGGCCAACACACCTATGGACC

GTCTTGCTGAACAATTTGCGAACCGCGAGTGTTCGACGTTG

TATGACCCTGAAAAAAAACGTGGACAGTATATTGTCGAGA

TGGCAAAAGAGCGTAAGGCCGACGGAATCATCTTCTTCAT

GACAAAATTCTGCGATCCCGAAGAATACGATTACCCTCAG

ATGAAAAAAGACTTCGAAGAAGCCGGTATTCCCCACGTTC

TGATTGAGACAGACATGCAAATGAAGAACTACGAACAAGC

TCGCACCGCTATTCAAGCATTTTCAGAAACCCTTTG

Acul ATGCGTGCTGTCTTAATCGAGAAGTCAGATGACACCCAGA

GTGTTTCAGTTACGGAGTTGGCTGAAGACCAATTACCCGAA

SEQ ID NO: 190 GGTGACGTCCTTGTGGATGTCGCGTACAGCACATTGAATTA

CAAGGATGCTCTTGCGATTACTGGAAAAGCACCCGTTGTAC

GCCGTTTTCCTATGGTCCCCGGAATTGACTTTACTGGGACT

GTCGCACAGAGTTCCCATGCTGATTTCAAGCCAGGCGACC

GCGTAATTCTGAACGGATGGGGAGTTGGTGAGAAACACTG

GGGCGGTCTTGCAGAACGCGCACGCGTACGTGGGGACTGG

CTTGTCCCGTTGCCAGCCCCCTTAGACTTGCGCCAGGCTGC

AATGATTGGCACTGCGGGGTACACAGCTATGCTGTGCGTG

CTTGCCCTTGAGCGCCATGGAGTCGTACCTGGGAACGGCG

AGATTGTCGTCTCAGGCGCAGCAGGAGGGGTAGGTTCTGT

AGCAACCACACTGTTAGCAGCCAAAGGCTACGAAGTGGCC

GCCGTGACCGGGCGCGCAAGCGAGGCCGAATATTTACGCG

GATTAGGCGCCGCGTCGGTCATTGATCGCAATGAATTAAC

GGGGAAGGTGCGTCCATTAGGGCAGGAACGCTGGGCAGGA

GGAATCGATGTAGCAGGATCAACCGTACTTGCTAATATGTT

GAGCATGATGAAATACCGTGGCGTGGTGGCGGCCTGTGGC

CTGGCGGCTGGAATGGACTTGCCCGCGTCTGTCGCCCCTTT

TATTCTGCGTGGTATGACTTTGGCAGGGGTAGATTCAGTCA

TGTGCCCCAAAACTGATCGTCTGGCTGCTTGGGCACGCCTG

GCATCCGACCTGGACCCTGCAAAGCTGGAAGAGATGACAA

CTGAATTACCGTTCTCTGAGGTGATTGAAACGGCTCCGAAG

TTCTTGGATGGAACAGTGCGTGGGCGTATTGTCATTCCGGT

AACACCTTGA

fldHl ATGAAAATCTTGGCATACTGCGTCCGCCCAGACGAGGTAG

ACTCCTTTAAGAAATTTAGTGAAAAGTACGGGCATACAGTT

SEQ ID NO: 191 GATCTTATTCCAGACTCTTTTGGACCTAATGTCGCTCATTTG

GCGAAGGGTTACGATGGGATTTCTATTCTGGGCAACGACA

CGTGTAACCGTGAGGCACTGGAGAAGATCAAGGATTGCGG

GATCAAATATCTGGCAACCCGTACAGCCGGAGTGAACAAC

ATTGACTTCGATGCAGCAAAGGAGTTCGGTATTAACGTGG

CTAATGTTCCCGCATATTCCCCCAACTCGGTCAGCGAATTT

ACCATTGGATTGGCATTAAGTCTGACGCGTAAGATTCCATT

TGCCCTGAAACGCGTGGAACTGAACAATTTTGCGCTTGGCG

GCCTTATTGGTGTGGAATTGCGTAACTTAACTTTAGGAGTC ATCGGTACTGGTCGCATCGGATTGAAAGTGATTGAGGGCTT

CTCTGGGTTTGGAATGAAAAAAATGATCGGTTATGACATTT

TTGAAAATGAAGAAGCAAAGAAGTACATCGAATACAAATC

ATTAGACGAAGTTTTTAAAGAGGCTGATATTATCACTCTGC

ATGCGCCTCTGACAGACGACAACTATCATATGATTGGTAA

AGAATCCATTGCTAAAATGAAGGATGGGGTATTTATTATCA

ACGCAGCGCGTGGAGCCTTAATCGATAGTGAGGCCCTGAT

TGAAGGGTTAAAATCGGGGAAGATTGCGGGCGCGGCTCTG

GATAGCTATGAGTATGAGCAAGGTGTCTTTCACAACAATA

AGATGAATGAAATTATGCAGGATGATACCTTGGAACGTCT

GAAATCTTTTCCCAACGTCGTGATCACGCCGCATTTGGGTT

TTTATACTGATGAGGCGGTTTCCAATATGGTAGAGATCACA

CTGATGAACCTTCAGGAATTCGAGTTGAAAGGAACCTGTA

AGAACCAGCGTGTTTGTAAATGA

FldD ATGTTCTTTACGGAGCAACACGAACTTATTCGCAAACTGGC

GCGTGACTTTGCCGAACAGGAAATCGAGCCTATCGCAGAC

SEQ ID NO: 193 GAAGTAGATAAAACCGCAGAGTTCCCAAAAGAAATCGTGA

AGAAGATGGCTCAAAATGGATTTTTCGGCATTAAAATGCCT

AAAGAATACGGAGGGGCGGGTGCGGATAACCGCGCTTATG

TCACTATTATGGAGGAAATTTCACGTGCTTCCGGGGTAGCG

GGTATCTACCTGAGCTCGCCGAACAGTTTGTTAGGAACTCC

CTTCTTATTGGTCGGAACCGATGAGCAAAAAGAAAAGTAC

CTTAAGCCTATGATCCGCGGCGAGAAGACTCTGGCGTTCGC

CCTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTGCGTTGG

CTACTACTGCCCGTGAAGAGGGCGACTATTATATCTTAAAT

GGCCGCAAGACGTTTATTACAGGGGCTCCTATTAGCGACA

ATATTATTGTGTTCGCAAAAACCGATATGAGCAAAGGGAC

CAAAGGTATCACCACTTTCATTGTGGACTCAAAGCAGGAA

GGGGTAAGTTTTGGTAAGCCAGAGGACAAAATGGGAATGA

TTGGTTGTCCGACAAGCGACATCATCTTGGAAAACGTTAAA

GTTCATAAGTCCGACATCTTGGGAGAAGTCAATAAGGGGT

TTATTACCGCGATGAAAACACTTTCCGTTGGTCGTATCGGA

GTGGCGTCACAGGCGCTTGGAATTGCACAGGCCGCCGTAG

ATGAGGCGGTAAAGTACGCCAAGCAACGTAAACAATTCAA

TCGCCCAATCGCGAAATTTCAGGCCATTCAATTTAAACTTG

CCAATATGGAGACTAAATTAAATGCCGCTAAACTTCTTGTT

TATAACGCAGCGTACAAAATGGATTGTGGAGAAAAAGCCG

ACAAGGAAGCCTCTATGGCTAAATACTTTGCTGCTGAATCA

GCGATCCAAATCGTTAACGACGCGCTGCAAATCCATGGCG

GGTATGGCTATATCAAAGACTACAAGATTGAACGTTTGTAC

CGCGATGTGCGTGTGATCGCTATTTATGAGGGCACTTCCGA

GGTCCAACAGATGGTTATCGCGTCCAATCTGCTGAAGTAA

[001] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more Trp aminotransferase. Accordingly, In some embodiments, the nucleic acid sequence comprising the Trp

aminotransferase gene has at least about 80% identity with SEQ ID NO: 95. In some embodiments, the gene sequence comprising the Trp aminotransferase gene has at least about 90% identity with SEQ ID NO: 95. In another embodiment, the gene sequence comprising the Trp aminotransferase gene has at least about 95% identity with SEQ ID NO: 95. Accordingly, In some embodiments, the gene sequence comprising the Trp aminotransferase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95. In another embodiment, the gene sequence comprising the Trp

aminotransferase gene comprises SEQ ID NO: 95. In yet another embodiment the gene sequence comprising the Trp aminotransferase gene consists of SEQ ID NO: 95.

[0656] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more Tryptophan Decarboxylase

Accordingly, In some embodiments, the nucleic acid sequence comprising the

Tryptophan Decarboxylase gene has at least about 80% identity with SEQ ID NO: 96. In some embodiments, the gene sequence comprising the Tryptophan Decarboxylase gene has at least about 90% identity with SEQ ID NO: 96. In another embodiment, the gene sequence comprising the Tryptophan Decarboxylase gene has at least about 95% identity with SEQ ID NO: 96. Accordingly, In some embodiments, the gene sequence comprising the Tryptophan Decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96. In another embodiment, the gene sequence comprising the

Tryptophan Decarboxylase gene comprises SEQ ID NO: 96. In yet another

embodiment the gene sequence comprising the Tryptophan Decarboxylase gene consists of SEQ ID NO: 96.

[0657] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 28. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 28. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 28. In some

embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 28. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 28. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 28. In another embodiment, the genetically engineered bacteria comprise the sequence of Table 28. In some embodiments, the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 28.

[0658] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tryptophan amino transferase. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tdc from C. roseus. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 95. In another embodiment, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 85% identity with SEQ ID NO: 95. In some embodiments, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 90% identity with SEQ ID NO: 95. In some embodiments, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 95% identity with SEQ ID NO: 95. In another embodiment, the tryptophan amino transferase gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95. Accordingly, In some embodiments, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95. In another embodiment, the genetically engineered bacteria comprise the tryptophan amino transferase gene sequence of SEQ ID NO: 95. In yet another embodiment the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which consists of the sequence of SEQ ID NO: 95.

[0659] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tryptophan decarboxylase. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tdc from C. roseus. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 96. In another embodiment, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 85% identity with SEQ ID NO: 96. In some embodiments, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 90% identity with SEQ ID NO: 96. In some embodiments, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 95% identity with SEQ ID NO: 96. In another embodiment, the tryptophan decarboxylase gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96. Accordingly, In some embodiments, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96. In another embodiment, the genetically engineered bacteria comprise the tryptophan decarboxylase gene sequence of SEQ ID NO: 96. In yet another embodiment the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which consists of the sequence of SEQ ID NO: 96.

[0660] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding Tdc. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tdc from C. roseus. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 171. In another embodiment, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 85% identity with SEQ ID NO: 171. In some embodiments, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 90% identity with SEQ ID NO: 171. In some embodiments, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 95% identity with SEQ ID NO: 171. In another embodiment, the Tdc gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 171. Accordingly, In some embodiments, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 171. In another embodiment, the genetically engineered bacteria comprise the Tdc gene sequence of SEQ ID NO: 171. In yet another embodiment the genetically engineered bacteria comprise a Tdc gene sequence which consists of the sequence of SEQ ID NO: 171. [0661] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tryptophan dehydrogenase (trpDH). In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 80% identity with SEQ ID NO: 175. In another embodiment, the genetically engineered bacteria comprise a trpDH sequence which has at least about 85% identity with SEQ ID NO: 175. In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 90% identity with SEQ ID NO: 175. In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 95% identity with SEQ ID NO: 175. In another embodiment, the a trpDH gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 175. Accordingly, In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 175. In another embodiment, the genetically engineered bacteria comprise the a trpDH gene sequence of SEQ ID NO: 175. In yet another embodiment the genetically engineered bacteria comprise a trpDH gene sequence which consists of the sequence of SEQ ID NO: 175.

[0662] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding ipdC. In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 80% identity with SEQ ID NO: 176. In another embodiment, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 85% identity with SEQ ID NO: 176. In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 90% identity with SEQ ID NO: 176. In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 95% identity with SEQ ID NO: 176. In another embodiment, the ipdC gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 176. Accordingly, In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 176. In another embodiment, the genetically engineered bacteria comprise the ipdC gene sequence of SEQ ID NO: 176. In yet another embodiment the genetically engineered bacteria comprise a ipdC gene sequence which consists of the sequence of SEQ ID NO: 176.

[0663] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding ladl. In some embodiments, the genetically engineered bacteria comprise a ladl gene sequence which has at least about 80% identity with SEQ ID NO: 177. In another embodiment, the genetically engineered bacteria comprise a ladl gene sequence which has at least about 85% identity with SEQ ID NO: 177. In some embodiments, the genetically engineered bacteria comprise a ladl gene sequence which has at least about 90% identity with SEQ ID NO: 177. In some embodiments, the genetically engineered bacteria comprise a ladl gene sequence which has at least about 95% identity with SEQ ID NO: 177. In another embodiment, the ladl gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 177. Accordingly, In some embodiments, the genetically engineered bacteria comprise a ladl gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 177. In another embodiment, the genetically engineered bacteria comprise the ladl gene sequence of SEQ ID NO: 177. In yet another embodiment the genetically engineered bacteria comprise a ladl gene sequence which consists of the sequence of SEQ ID NO: 177.

[0664]

[0665] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldA. In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 80% identity with SEQ ID NO: 187. In another embodiment, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 85% identity with SEQ ID NO: 187. In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 90% identity with SEQ ID NO: 187. In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 95% identity with SEQ ID NO: 187. In another embodiment, the fldA gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 187. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 187. In another embodiment, the genetically engineered bacteria comprise the fldA gene sequence of SEQ ID NO: 187. In yet another embodiment the genetically engineered bacteria comprise a fldA gene sequence which consists of the sequence of SEQ ID NO: 187.

[0666] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldB. In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 80% identity with SEQ ID NO: 188. In another embodiment, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 85% identity with SEQ ID NO: 188. In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 90% identity with SEQ ID NO: 188. In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 95% identity with SEQ ID NO: 188. In another embodiment, the fldB gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 188. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 188. In another embodiment, the genetically engineered bacteria comprise the fldB gene sequence of SEQ ID NO: 188. In yet another embodiment the genetically engineered bacteria comprise a fldB gene sequence which consists of the sequence of SEQ ID NO: 188.

[0667] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldC. In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 80% identity with SEQ ID NO: 189. In another embodiment, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 85% identity with SEQ ID NO: 189. In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 90% identity with SEQ ID NO: 189. In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 95% identity with SEQ ID NO: 189. In another embodiment, the fldC gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 189. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 189. In another embodiment, the genetically engineered bacteria comprise the fldC gene sequence of SEQ ID NO: 189. In yet another embodiment the genetically engineered bacteria comprise a fldC gene sequence which consists of the sequence of SEQ ID NO: 189.

[0668] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding Acul. In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 80% identity with SEQ ID NO: 190. In another embodiment, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 85% identity with SEQ ID NO: 190. In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 90% identity with SEQ ID NO: 190. In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 95% identity with SEQ ID NO: 190. In another embodiment, the Acul gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 190. Accordingly, In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 190. In another embodiment, the genetically engineered bacteria comprise the Acul gene sequence of SEQ ID NO: 190. In yet another embodiment the genetically engineered bacteria comprise a Acul gene sequence which consists of the sequence of SEQ ID NO: 190.

[0669] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldHl. In some embodiments, the genetically engineered bacteria comprise a fldHl gene sequence which has at least about 80% identity with SEQ ID NO: 191. In another embodiment, the genetically engineered bacteria comprise a fldHl gene sequence which has at least about 85% identity with SEQ ID NO: 191. In some embodiments, the genetically engineered bacteria comprise a fldHl gene sequence which has at least about 90% identity with SEQ ID NO: 191. In some embodiments, the genetically engineered bacteria comprise a fldHl gene sequence which has at least about 95% identity with SEQ ID NO: 191. In another embodiment, the fldHl gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 191. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldHl gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 191. In another embodiment, the genetically engineered bacteria comprise the fldHl gene sequence of SEQ ID NO: 191. In yet another embodiment the genetically engineered bacteria comprise a fldHl gene sequence which consists of the sequence of SEQ ID NO: 191.

[0670] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldD. In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 80% identity with SEQ ID NO: 193. In another embodiment, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 85% identity with SEQ ID NO: 193. In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 90% identity with SEQ ID NO: 193. In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 95% identity with SEQ ID NO: 193. In another embodiment, the fldD gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 193. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 193. In another embodiment, the genetically engineered bacteria comprise the fldD gene sequence of SEQ ID NO: 193. In yet another embodiment the genetically engineered bacteria comprise a fldD gene sequence which consists of the sequence of SEQ ID NO: 193.

Table 29. Tryptophan Pathway Catabolic Enzymes

FRLKPDVSSLHVEEVNKKLLDMLNSTGRVYMTHTIVGGIYML RLAVGSSLTEEHHVRRVWDLIQKLTDDLLKEA

TDC: Tryptophan MKFWRKYTQQEMDEKITESLEKTLNYDNTKTIGIPGTKLDDT decarboxylase from VFYDDHSFVKHSPYLRTFIQNPNHIGCHTYDKADILFGGTFDIE Clostridium RELIQLLAIDVLNGNDEEFDGYVTQGGTEANIQAMWVYRNY sporogenes FKKERKAKHEEIAIITSADTHYSAYKGSDLLNIDIIKVPVDFYS SEQ ID NO: 98 RKIQENTLDSIVKEAKEIGKKYFIVISNMGTTMFGSVDDPDLY

ANIFDKYNLEYKIHVDGAFGGFIYPIDNKECKTDFSNKNVSSIT

LDGHKMLQAPYGTGIFVSRKNLIHNTLTKEATYIENLDVTLSG

S RS GS N A V AI WM VL AS YGP YG WME KINKLRNRTKWLC KQL

NDMRIKYYKEDSMNIVTIEEQYVNKEIAEKYFLVPEVHNPTN

NWYKIVVMEHVELDILNSLVYDLRKFNKEHLKAM

Tryptophan MSQVIKKKRNTFMIGTEYILNSTQLEEAIKSFVHDFCAEKHEIH Decarboxylase (EC DQPVVVEAKEHQEDKIKQIKIPEKGRPVNEVVSEMMNEVYRY 4.1.1.28) Chain A, RGD ANHPRFFS FVPGP AS S VS WLGDIMTS A YNIH AGGS KL AP Ruminococcus MVNCIEQEVLKWLAKQVGFTENPGGVFVSGGSMANITALTA Gnavus Tryptophan ARDNKLTDINLHLGTAYISDQTHSSVAKGLRIIGITDSRIRRIPT

NSHFQMDTTKLEEAIETDKKSGYIPFVVIGTAGTTNTGSIDPLT

SEQ ID NO: 99 EISALCKKHDMWFHIDGAYGASVLLSPKYKSLLTGTGLADSIS

WDAHKWLFQTYGCAMVLVKDIRNLFHSFHVNPEYLKDLEN

DIDN VNT WDIGMELTRP ARGLKLWLTLQ VLGS DLIGS AIEHG

FQLAVWAEEALNPKKDWEIVSPAQMAMINFRYAPKDLTKEE

QDILNEKISHRILESGYAAIFTTVLNGKTVLRICAIHPEATQED

MQHTIDLLDQYGREIYTEMKKa

Trp MTATTISIETVPQAPAAGTKTNGTSGKYNPRTYLSDRAKVTEI aminotransferase DGS D AGRPNPDTFPFNS ITLNLKPPLGLPES S NNMP VS ITIEDPD (EC 2.6.1.27); L AT ALQ Y APS AGIPKLRE WL ADLQ AH VHERPRGD Y AIS VGS G tryptophan S QDLMFKGFQ A VLNPGDP VLLETPM YS G VLP ALRILKAD Y AE aminotransferase VD VDDQGLS AKNLE KVLS E WP AD KKRPR VL YTS PIGS NPS GC [Cryptococcus S AS KERKLE VLKVC KKYD VLIFEDDP Y Y YL AQELIPS YFALE K deuterogattii R265] QVYPEGGHVVRFDSFSKLLSAGMRLGFATGPKEILHAIDVSTA SEQ ID NO: 100 GANLHTSAVSQGVALRLMQYWGIEGFLAHGRAVAKLYTERR

AQFEATAHKYLDGLATWVSPVAGMFLWIDLRPAGIEDSYELI RHE AL AKG VLG VPGM AFYPTGRKS S H VRVS FS I VDLEDES DL GFQRLAEAIKDKRKALGLA

AR09: L- tryptophan MT AGS APP VD YTS LKKNFQPFLS RRVENRS LKS FWD AS DIS D aminotransferase DVIELAGGMPNERFFPIESMDLKISKVPFNDNPKWHNSFTTAH from S. cerevisae LDLGS PS ELPI ARS FQ Y AET KGLPPLLHFVKDFVS RINRP AFS D SEQ ID NO: 103 ETES NWD VILS GGS NDS MFKVFETICDES TT VMIEEFTFTP AM

SNVEATGAKVIPIKMNLTFDRESQGIDVEYLTQLLDNWSTGP YKDLNKPRVLYTIATGQNPTGMSVPQWKREKIYQLAQRHDF LIVEDDPYGYLYFPSYNPQEPLENPYHSSDLTTERYLNDFLMK SFLTLDTDARVIRLETFSKIFAPGLRLSFIVANKFLLQKILDLAD ITTRAPSGTSQAIVYSTIKAMAESNLSSSLSMKEAMFEGWIRW IMQIASKYNHRKNLTLKALYETESYQAGQFTVMEPSAGMFIII KINWGNFDRPDDLPQQMDILDKFLLKNGVKVVLGYKMAVCP NYSKQNSDFLRLTIAYARDDDQLIEASKRIGSGIKEFFDNYKS

TYNA: Monoamine MGS PS LYS ARKTTLAL AVALS F A WQ AP VF AHGGE AHM VPM oxidase from E. coli DKTLKEFGADVQWDDYAQLFTLIKDGAYVKVKPGAQTAIVN SEQ ID NO: 101 GQPLALQVPVVMKDNKAWVSDTFINDVFQSGLDQTFQVEKR

PHPLNALTADEIKQAVEIVKASADFKPNTRFTEISLLPPDKEAV

WAFALENKPVDQPRKADVIMLDGKHIIEAVVDLQNNKLLSW

QPIKDAHGMVLLDDFASVQNIINNSEEFAAAVKKRGITDAKK

VITTPLTVGYFDGKDGLKQDARLLKVISYLDVGDGNYWAHPI

ENLVAVVDLEQKKIVKIEEGPVVPVPMTARPFDGRDRVAPAV

KPMQIIEPEGKNYTITGDMIHWRNWDFHLSMNSRVGPMISTV

TYNDNGTKRKVMYEGSLGGMIVPYGDPDIGWYFKAYLDSGD

YGMGTLTSPIARGKDAPSNAVLLNETIADYTGVPMEIPRAIAV

FERYAGPEYKHQEMGQPNVSTERRELVVRWISTVGNYDYIFD

WIFHENGTIGIDAGATGIEAVKGVKAKTMHDETAKDDTRYGT

LIDHNIVGTTHQHIYNFRLDLDVDGENNSLVAMDPVVKPNTA

GGPRTSTMQVNQYNIGNEQDAAQKFDPGTIRLLSNPNKENRM

GNP VS YQIIP Y AGGTHP V AKG AQF APDE WI YHRLS FMD KQLW

VTRYHPGERFPEGKYPNRSTHDTGLGQYSKDNESLDNTDAV

VWMTTGTTHVARAEEWPIMPTEWVHTLLKPWNFFDETPTLG

ALKKDK

AAOl: Indole-3- MGEKAIDEDKVEAMKSSKTSLVFAINGQRFELELSSIDPSTTL acetaldehyde oxidase VDFLRNKTPFKSVKLGCGEGGCGACVVLLSKYDPLLEKVDEF from Arabidopsis TISSCLTLLCSIDGCSITTSDGLGNSRVGFHAVHERIAGFHATQ thaliana CGFCTPGMS VS MFS ALLN AD KS HPPPRS GFS NLT A VE AE KA V

SEQ ID NO: 102 S GNLCRCTG YRPLVD AC KS F A AD VDIEDLGFN AFC KKGENRD

E VLRRLPC YDHTS S H VCTFPEFLKKEIKNDMS LHS RKYRWS S P

VSVSELQGLLEVENGLSVKLVAGNTSTGYYKEEKERKYERFI

DIRKIPEFTM VRS DE KG VELG AC VTIS KAIE VLREEKN VS VL A

KIATHMEKIANRFVRNTGTIGGNIMMAQRKQFPSDLATILVA

AQ AT VKIMTS S S S QEQFTLEEFLQQPPLD AKS LLLSLEIPS WHS

AKKNGSSEDSILLFETYRAAPRPLGNALAFLNAAFSAEVTEAL

DGIVVNDCQLVFGAYGTKHAHRAKKVEEFLTGKVISDEVLM

E AIS LLKDEI VPD KGTS NPG YRS S L A VTFLFEFFGS LT KKN AKT

TNGWLNGGC KEIGFD QN VES LKPE AMLS S AQQI VENQEHS P V

GKGITKAGACLQASGEAVYVDDIPAPENCLYGAFIYSTMPLA

RIKGIRFKQNRVPEGVLGIITYKDIPKGGQNIGTNGFFTSDLLF

AEEVTHCAGQIIAFLVADSQKHADIAANLVVIDYDTKDLKPPI

LSLEEAVENFSLFEVPPPLRGYPVGDITKGMDEAEHKILGSKIS

FGS Q YFFYMETQT AL A VPDEDNCM V V YS S T QTPEFVHQTI AG

CLGVPENNVRVITRRVGGGFGGKAVKSMPVAAACALAASK

MQRPVRTYVNRKTDMITTGGRHPMKVTYSVGFKSNGKITAL

DVEVLLDAGLTEDISPLMPKGIQGALMKYDWGALSFNVKVC

KTNT VS RT ALR APGD VQGS YIGE AIIEKV AS YLS VD VDEIRKV

NLHTYESLRLFHSAKAGEFSEYTLPLLWDRIDEFSGFNKRRKV

VEEFN AS NKWRKRGIS RVP A V Y A VNMRS TPGRVS VLGDGS IV

VEVQGIEIGQGLWTKVKQMAAYSLGLIQCGTTSDELLKKIRVI QS DTLS M VQGS MT AGS TTS E AS S E A VRICCD GLVERLLP VKT

ALVEQTGGPVTWDSLISQAYQQSINMSVSSKYMPDSTGEYLN

YGIAASEVEVNVLTGETTILRTDIIYDCGKSLNPAVDLGQIEGA

FVQGLGFFMLEEFLMNSDGLVVTDSTWTYKIPTVDTIPRQFN

VEILNS GQHKNRVLS S KAS GEPPLLLAAS VHC A VRAAVKE AR

KQILSWNSNKQGTDMYFELPVPATMPIVKEFCGLDVVEKYLE

WKIQQRKNV

aspC: aspartate MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKT aminotransferase PVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKG from E. coli S ALIND KR ART AQTPGGTG ALRV A ADFL AKNTS VKRV WVS N SEQ ID NO: 104 PSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEA

QAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLF

DFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYN

ERVG ACTLV A ADS ET VDR AFS QMKA AIR AN YS NPP AHG AS V

VATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGAN

RDFS FIIKQNGMFS FS GLT KEQ VLRLREEFG V Y A V AS GRVN V A

GMTPDNMAPLCEAIVAVL

TAA1: L- tryptophan- M VKLENS RKPE KIS NKNIPMS DFV VNLDHGDPT A YEE YWRK pyruvate MGDRCTVTIRGCDLMSYFSDMTNLCWFLEPELEDAIKDLHGV aminotransferase VGNAATEDRYIVVGTGSTQLCQ AAVH ALS S LARS QPVS VV A from Arabidopsis AAPFYSTYVEETTYVRSGMYKWEGDAWGFDKKGPYIELVTS thaliana PNNPDGTIRETVVNRPDDDEAKVIHDFAYYWPHYTPITRRQD

SEQ ID NO: 105 HDIMLFTFSKITGHAGSRIGWALVKDKEVAKKMVEYIIVNSIG

VSKESQVRTAKILNVLKETCKSESESENFFKYGREMMKNRWE

KLREVVKESDAFTLPKYPEAFCNYFGKSLESYPAFAWLGTKE

ETDLVS ELRRHKVMS R AGERCGS D KKH VR VS MLS RED VFN V

FLERLANMKLIKSIDL

STAO: L-tryptophan MTAPLQDSDGPDDAIGGPKQVTVIGAGIAGLVTAYELERLGH oxidase from HVQIIEGSDDIGGRIHTHRFSGAGGPGPFAEMGAMRIPAGHRL streptomyces sp. TP- TMHYIAELGLQNQVREFRTLFSDDAAYLPSSAGYLRVREAHD A0274 TLVDEFATGLPSAHYRQDTLLFGAWLDASIRAIAPRQFYDGL

SEQ ID NO: 106 HNDIGVELLNLVDDIDLTPYRCGTARNRIDLHALFADHPRVR

ASCPPRLERFLDDVLDETSSSIVRLKDGMDELPRRLASRIRGKI

SLGQEVTGIDVHDDTVTLTVRQGLRTVTRTCDYVVCTIPFTVL

RTLRLTGFDQDKLDIVHETKYWPATKIAFHCREPFWEKDGIS

GGASFTGGHVRQTYYPPAEGDPALGAVLLASYTIGPDAEALA

RMDEAERDALVAKELSVMHPELRRPGMVLAVAGRDWGARR

WSRGAATVRWGQEAALREAERRECARPQKGLFFAGEHCSSK

P A WIEG AIES AID A AHEIE WYEPRAS RVF A AS RLS RS DRS A

ipdC: Indole- 3- MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVIDS pyruvate PDICWVGCANELNASYAADGYARCKGFAALLTTFGVGELSA decarboxylase from MNGIAGSYAEHVPVLHIVGAPGTAAQQRGELLHHTLGDGEFR Enterobacter cloacae HFYHMSEPITVAQAVLTEQNACYEIDRVLTTMLRERRPGYLM SEQ ID NO: 107 LPADVAKKAATPPVNALTHKQAHADSACLKAFRDAAENKLA

MSKRTALLADFLVLRHGLKHALQKWVKEVPMAHATMLMG

KGIFDERQAGFYGTYSGSASTGAVKEAIEGADTVLCVGTRFT

DTLTAGFTHQLTPAQTIEVQPHAARVGDVWFTGIPMNQAIET

LVELCKQHVH AGLMS S S S GAIPFPQPDGS LTQENFWRTLQTFI

RPGDIILADQGTSAFGAIDLRLPADVNFIVQPLWGSIGYTLAA AFGAQTACPNRRVIVLTGDGAAQLTIQELGSMLRDKQHPIILV LNNEGYTVERAIHGAEQRYNDIALWNWTHIPQALSLDPQSEC WRVSEAEQLADVLEKVAHHERLSLIEVMLPKADIPPLLGALT KALEACNNA

IAD1: Indole-3- MPTLNLDLPNGIKSTIQADLFINNKFVPALDGKTFATINPSTGK acetaldehyde EIGQVAEASAKDVDLAVKAAREAFETTWGENTPGDARGRLLI dehydrogenase from KLAELVEANIDELAAIESLDNGKAFSIAKSFDVAAVAANLRY Ustilago maydis YGGWADKNHGKVMEVDTKRLNYTRHEPIGVCGQIIPWNFPL SEQ ID NO: 108 LMFAWKLGPALATGNTIVLKTAEQTPLSAIKMCELIVEAGFPP

GVVNVISGFGPVAGAAISQHMDIDKIAFTGSTLVGRNIMKAA

ASTNLKKVTLELGGKSPNIIFKDADLDQAVRWSAFGIMFNHG

QCCC AGS R V Y VEES I YD AFME KMT AHC KALQ VGDPFS ANTF

QGPQVS QLQYDRIME YIES GKKD ANLALGG VRKGNEGYFIEP

TIFTDVPHDAKIAKEEIFGPVVVVSKFKDEKDLIRIANDSIYGL

AAAVFSRDISRAIETAHKLKAGTVWVNCYNQLIPQVPFGGYK

AS GIGRELGE Y ALS N YTNIKA VH VNLS QP API

YUC2: indole-3- MEFVTETLGKRIHDPYVEETRCLMIPGPIIVGS GPS GLAT AACL pyruvate KS RDIPS LILERS TCI AS LWQHKT YDRLRLHLPKDFCELPLMPF monoxygenase from PSSYPTYPTKQQFVQYLESYAEHFDLKPVFNQTVEEAKFDRR Arabidopsis thaliana CGLWRVRTTGGKKDETMEYVSRWLVVATGENAEEVMPEID SEQ ID NO: 109 GIPDFGGPILHTS S YKS GEIFSEKKILVVGCGNS GME VCLDLCN

FN ALPS LV VRDS VH VLPQEMLGIS TFGIS TS LLKWFP VH V VDR FLLRMSRLVLGDTDRLGLVRPKLGPLERKIKCGKTPVLDVGT LAKIRS GHIKVYPELKRVMHYS AEFVDGRVDNFD AIILATGY KSNVPMWLKGVNMFSEKDGFPHKPFPNGWKGESGLYAVGF TKLGLLGAAIDAKKIAEDIEVQRHFLPLARPQHC

IaaM: Tryptophan 2- M YDHFNS PS IDILYD YGPFLKKCEMTGGIGS YS AGTPTPRV AI monooxygenase from VGAGISGLVAATELLRAGVKDVVLYESRDRIGGRVWSQVFD

Pseudomonas QTRPRYIAEMGAMRFPPSATGLFHYLKKFGISTSTTFPDPGVV savastanoi DTELHYRGKRYHWPAGKKPPELFRRVYEGWQSLLSEGYLLE

SEQ ID NO: 110 GGSLVAPLDITAMLKSGRLEEAAIAWQGWLNVFRDCSFYNAI

VCIFTGRHPPGGDRWARPEDFELFGSLGIGSGGFLPVFQAGFT

EILRMVINGYQSDQRLIPDGISSLAARLADQSFDGKALRDRVC

FS RVGRIS RE AE KIIIQTE AGEQRVFDRVI VTS S NRAMQMIHCL

TDSESFLSRDVARAVRETHLTGSSKLFILTRTKFWIKNKLPTTI

QSDGLVRGVYCLDYQPDEPEGHGVVLLSYTWEDDAQKMLA

MPDKKTRCQVLVDDLAAIHPTFASYLLPVDGDYERYVLHHD

WLTDPHSAGAFKLNYPGEDVYSQRLFFQPMTANSPNKDTGL

YLAGCSCS FAGGWIEG AVQT ALNS AC AVLRSTGGQLS KGNPL

DCINASYRY

iaaH: MHEIITLESLCQALADGEIAAAELRERALDTEARLARLNCFIRE

Indo leacetamide GD AVS QFGE ADH AMKGTPLWGMPVS FKDNIC VRGLPLT AGT hydrolase from RGMSGFVSDQDAAIVSQLRALGAVVAGKNNMHELSFGVTSI

Pseudomonas NPHWGT VGNP V APG YC AGGS S GGS A AAV AS GI VPLS VGTDT savastanoi GGSIRIPAAFCGITGFRPTTGRWSTAGIIPVSHTKDCVGLLTRT

SEQ ID NO: 111 AGDAGFLYGLLSGKQQSFPLSRTAPCRIGLPVSMWSDLDGEV

ERACVNALSLLRKTGFEFIEIDDADIVELNQTLTFTVPLYEFFA DLAQSLLSLGWKHGIHHIFAQVDDANVKGIINHHLGEGAIKP AH YLS S LQNGELLKRKMDELF ARHNIELLG YPT VPCRVPHLD HADRPEFFSQAIRNTDLASNAMLPSITIPVGPEGRLPVGLSFDA LRGRDALLLSRVSAIEQVLGFVRKVLPHTT

TrpDH: Tryptophan MLLFETVREMGHEQVLFCHSKNPEIKAIIAIHDTTLGPAMGAT dehydrogenase from RILPYINEEAALKDALRLSRGMTYKAACANIPAGGGKAVIIAN Nostoc punctiforme PENKTDDLLRAYGRFVDS LNGRFITGQD VNITPDD VRTIS QET NIES-2108 KYVVGVSEKSGGPAPITSLGVFLGIKAAVESRWQSKRLDGMK SEQ ID NO: 112 VAVQGLGNVGKNLCRHLHEHDVQLFVSDVDPIKAEEVKRLF

G AT V VEPTEI YS LD VDIFAPC ALGGILNS HTIPFLQ AS II AG A AN

NQLENEQLHSQMLAKKGILYSPDYVINAGGLINVYNEMIGYD

EEKAFKQVHNIYDTLLAIFEIAKEQGVTTNDAARRLAEDRINN

SKRSKSKAIAA

CYP79B2: MNTFTS NS S DLTTT ATETS S FS TLYLLS TLQ AFV AITLVMLLKK tryptophan N- LMTDPNKKKPYLPPGPTGWPIIGMIPTMLKSRPVFRWLHSIMK monooxygenase from QLNTEIACVKLGNTHVITVTCPKIAREILKQQDALFASRPLTY Arabidopsis thaliana AQKILSNGYKTCVITPFGDQFKKMRKVVMTELVCPARHRWL SEQ ID NO: 113 HQKRSEENDHLTAWVYNMVKNSGSVDFRFMTRHYCGNAIK

KLMFGTRTFSKNTAPDGGPTVEDVEHMEAMFEALGFTFAFCI

S D YLPMLTGLDLNGHE KIMRES S AIMD KYHDPIIDERIKM WR

EGKRTQIEDFLDIFISIKDEQGNPLLTADEIKPTIKELVMAAPDN

PSNAVEWAMAEMVNKPEILRKAMEEIDRVVGKERLVQESDIP

KLNYVKAILREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGS

QVLLSRYGLGRNPKVWADPLCFKPERHLNECSEVTLTENDLR

FISFSTGKRGCAAPALGTALTTMMLARLLQGFTWKLPENETR

VELMESSHDMFLAKPLVMVGDLRLPEHLYPTVK

CYP79B3: MDTL AS NS S DLTTKS S LGMS S FTNM YLLTTLQ AL A ALCFLMI tryptophan N- LNKIKSSSRNKKLHPLPPGPTGFPIVGMIPAMLKNRPVFRWLH monooxygenase from SLMKELNTEIACVRLGNTHVIPVTCPKIAREIFKQQDALFASRP Arabidopsis thaliana LTYAQKILSNGYKTCVITPFGEQFKKMRKVIMTEIVCPARHR SEQ ID NO: 114 WLHDNRAEETDHLTAWLYNMVKNSEPVDLRFVTRHYCGNA

IKRLMFGTRTFSEKTEADGGPTLEDIEHMDAMFEGLGFTFAFC

IS D YLPMLTGLDLNGHE KIMRES S AIMD KYHDPIIDERIKM WR

EGKRTQIEDFLDIFISIKDEAGQPLLTADEIKPTIKELVMAAPDN

PSNAVEWAIAEMINKPEILHKAMEEIDRVVGKERFVQESDIPK

LNYVKAIIREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGSQV

LLS RYGLGRNPKV WS DPLS FKPERHLNECS E VTLTENDLRFIS

FSTGKRGCAAPALGTAITTMMLARLLQGFKWKLAGSETRVE

LMESSHDMFLSKPLVLVGELRLSEDLYPMVK

CYP71A13: MS NIQEMEMILS IS LCLTTLITLLLLRRFLKRT AT KVNLPPS P W indoleacetaldoxime RLP VIGNLHQLS LHPHRS LRS LS LRYGPLMLLHFGRVPIL V VS S dehydratase from GEAAQEVLKTHDHKFANRPRSKAVHGLMNGGRDVVFAPYG Arabidopis thaliana EYWRQMKSVCILNLLTNKMVESFEKVREDEVNAMIEKLEKA SEQ ID NO: 115 S S S S S SENLS ELFITLPS D VTS RV ALGRKHS EDET ARDLKKRVR

QIMELLGEFPIGEYVPILAWIDGIRGFNNKIKEVSRGFSDLMDK V VQEHLE AS ND KADFVDILLS IEKD KNS GFQ VQRNDIKFMILD MFIGGTS TTS TLLE WTMTELIRS PKS MKKLQDEIRS TIRPHGS Y IKEKEVENMKYLKAVIKEVLRLHPSLPMILPRLLSEDVKVKGY NIAAGTEVIINAWAIQRDTAIWGPDAEEFKPERHLDSGLDYHG KNLNYIPFGSGRRICPGINLALGLAEVTVANLVGRFDWRVEA GPNGDQPDLTEAIGIDVCRKFPLIAFPSSVV PEN2: myrosinase M AHLQRTFPTEMS KGRASFPKGFLFGT AS S S YQYEGAVNEG A from Arabidopsis RGQSVWDHFSNRFPHRISDSSDGNVAVDFYHRYKEDIKRMK thaliana DINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDELLA

SEQ ID NO: 116 NEITPLVTIFHWDIPQDLEDEYGGFLSEQIIDDFRDYASLCFERF

GDRVS LWCTMNEPWV YS V AG YDTGRKAPGRCS KY VNG AS V

AGMSGYEAYIVSHNMLLAHAEAVEVFRKCDHIKNGQIGIAHN

PLWYEPYDPSDPDDVEGCNRAMDFMLGWHQHPTACGDYPE

TMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSSLFVKSIKH

VDPTQPTWRTDQGVDWMKTNIDGKQIAKQGGSEWSFTYPTG

LRNILKYVKKTYGNPPILITENGYGEVAEQSQSLYMYNPSIDT

ERLE YIEGHIHAIHQAIHEDGVRVEGYYVWS LLDNFE WNS GY

GVRYGLYYIDYKDGLRRYPKMSALWLKEFLRFDQEDDSSTS

KKEEKKESYGKQLLHSVQDSQFVHSIKDSGALPAVLGSLFVV

SATVGTSLFFKGANN

Nitl: Nitrilase from MS S TKDMS T VQN ATPFNG V APS TT VRVTI VQS S T V YNDTP ATI Arabidopsis thaliana DKAEKYIVEAASKGAELVLFPEGFIGGYPRGFRFGLAVGVHN SEQ ID NO: 117 EEGRDEFRKYHASAIHVPGPEVARLADVARKNHVYLVMGAI

EKEGYTLYCTVLFFSPQGQFLGKHRKLMPTSLERCIWGQGDG

STIPVYDTPIGKLGAAICWENRMPLYRTALYAKGIELYCAPTA

DGSKEWQSSMLHIAIEGGCFVLSACQFCQRKHFPDHPDYLFT

D WYDD KEHDS I VS QGGS VIIS PLGQ VLAGPNFES EGLVT ADID

LGDIARAKLYFDSVGHYSRPDVLHLTVNEHPRKSVTFVTKVE

KAEDDSNK

IDOl: indoleamine M AH AMENS WTIS KE YHIDEE VGF ALPNPQENLPDFYND WMFI 2,3-dioxygenase from AKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLG homo sapiens CITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVY SEQ ID NO: 118 ADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVS

LLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKA

LQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGF

WEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQ

DMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDA

C VKALVS LRS YHLQIVTKYILIP AS QQPKENKTSEDPS KLE AK

GTGGTDLMNFLKTVRSTTEKSLLKEG

TD02: tryptophan MSGCPFLGNNFGYTFKKLPVEGSEEDKSQTGVNRASKGGLIY 2,3-dioxygenase from GNYLHLEKVLNAQELQSETKGNKIHDEHLFIITHQAYELWFK homo sapiens QILWELDS VREIFQNGH VRDERNMLKV VS RMHRVS VILKLLV SEQ ID NO: 119 QQFSILETMTALDFNDFREYLSPASGFQSLQFRLLENKIGVLQ

NMRVPYNRRHYRDNFKGEENELLLKSEQEKTLLELVEAWLE

RTPGLEPHGFNFWGKLEKNITRGLEEEFIRIQAKEESEEKEEQV

AEFQKQKEVLLSLFDEKRHEHLLSKGERRLSYRALQGALMIY

FYREEPRFQ VPFQLLTS LMDIDS LMT KWR YNH VCM VHRMLG

S KAGTGGS S G YH YLRS TVS DRYKVFVDLFNLS T YLIPRH WIPK

MNPTIHKFLYTAEYCDSSYFSSDESD

BNA2: indoleamine MNNTSITGPQVLHRTKMRPLPVLEKYCISPHHGFLDDRLPLTR 2,3-dioxygenase from LSSKKYMKWEEIVADLPSLLQEDNKVRSVIDGLDVLDLDETIL S. cerevisiae GD VRELRR A YS ILGFM AH A YI W AS GTPRD VLPECI ARPLLET A SEQ ID NO: 120 HILGVPPLATYSSLVLWNFKVTDECKKTETGCLDLENITTINTF

TGTVDESWFYLVSVRFEKIGSACLNHGLQILRAIRSGDKGDA

NVIDGLEGLAATIERLSKALMEMELKCEPNVFYFKIRPFLAGW TNMSHMGLPQGVRYGAEGQYRIFSGGSNAQSSLIQTLDILLG VKHT AN AAHS S QGDS KINYLDEMKKYMPREHREFLYHLES V CNIRE Y VS RN AS NR ALQE A YGRCIS MLKIFRDNHIQI VTKYIIL PSNSKQHGSNKPNVLSPIEPNTKASGCLGHKVASSKTIGTGGT RLMPFLKQCRDETVATADIKNEDKN

Afmid: Kynurenine M AFPS LS AGQNP WRNLS S EELEKQ YS PS RW VIHTKPEE V VGN formamidase from FVQIGSQATQKARATRRNQLDVPYGDGEGEKLDIYFPDEDSK mouse AFPLFLFLHGGYWQS GS KDDS AFMVNPLT AQGIVVVIVAYDI

SEQ ID NO: 121 APKGTLDQMVDQVTRSVVFLQRRYPSNEGIYLCGHSAGAHL

AAMVLLARWTKHGVTPNLQGFLLVSGIYDLEPLIATSQNDPL

RMTLEDAQRNSPQRHLDVVPAQPVAPACPVLVLVGQHDSPE

FHRQSKEFYETLLRVGWKASFQQLRGVDHFDIIENLTREDDV

LTQIILKTVFQKL

BNA3: kynurenine— MKQRFIRQFTNLMSTSRPKVVANKYFTSNTAKDVWSLTNEA oxoglutarate AAKAANNSKNQGRELINLGQGFFSYSPPQFAIKEAQKALDIPM transaminase from S. VNQ YS PTRGRPS LINS LIKLYS PI YNTELKAEN VT VTTG ANEGI cerevisae LSCLMGLLNAGDEVIVFEPFFDQYIPNIELCGGKVVYVPINPPK

SEQ ID NO: 122 ELDQRNTRGEEWTIDFEQFEKAITSKTKAVIINTPHNPIGKVFT

REELTTLGNICVKHNVVIISDEVYEHLYFTDSFTRIATLSPEIGQ

LTLTVGSAGKSFAATGWRIGWVLSLNAELLSYAAKAHTRICF

AS PS PLQE AC ANS IND ALKIG YFE KMRQE YINKFKIFTS IFDEL

GLPYTAPEGTYFVLVDFSKVKIPEDYPYPEEILNKGKDFRISH

WLINELGVVAIPPTEFYIKEHEKAAENLLRFAVCKDDAYLEN

AVERLKLLKDYL

GOT2: Aspartate M ALLHS GRVLPGI A A AFHPGL A A A AS AR AS S WWTH VEMGPP aminotransferase, DPILGVTEAFKRDTNSKKMNLGVGAYRDDNGKPYVLPSVRK mitochondrial from AE AQI A AKNLD KE YLPIGGL AEFC KAS AEL ALGENS E VLKS G homo sapiens RFVTVQTISGTGALRIGASFLQRFFKFSRDVFLPKPTWGNHTPI SEQ ID NO: 123 FRDAGMQLQGYRYYDPKTCGFDFTGAVEDISKIPEQSVLLLH

ACAHNPTGVDPRPEQWKEIATVVKKRNLFAFFDMAYQGFAS

GDGDKDAWAVRHFIEQGINVCLCQSYAKNMGLYGERVGAFT

MVCKDADEAKRVESQLKILIRPMYSNPPLNGARIAAAILNTPD

LRKQWLQEVKVMADRIIGMRTQLVSNLKKEGSTHNWQHITD

QIGMFCFTGLKPEQVERLIKEFSIYMTKDGRISVAGVTSSNVG

YLAHAIHQVTK

AADAT: MN Y ARFIT A AS A ARNPS PIRTMTDILS RGPKS MIS L AGGLPNP

Kynurenine/alpha- NMFPFKT A VIT VENGKTIQFGEEMMKR ALQ YS PS AGIPELLS W aminoadipate LKQLQIKLHNPPTIHYPPS QGQMDLC VTS GS QQGLCKVFEMII aminotransferase, NPGDN VLLDEP A YS GTLQS LHPLGCNIIN V AS DES GI VPDS LR mitochondrial DILSRWKPEDAKNPQKNTPKFLYTVPNGNNPTGNSLTSERKK SEQ ID NO: 124 EIYELARKYDFLIIEDDPYYFLQFNKFRVPTFLSMDVDGRVIRA

DS FS KIIS S GLRIGFLTGPKPLIERVILHIQ VS TLHPS TFNQLMIS QLLHEWGEEGFMAHVDRVIDFYSNQKDAILAAADKWLTGLA EWHVPAAGMFLWIKVKGINDVKELIEEKAVKMGVLMLPGN AFYVDS S APSPYLRAS FS S ASPEQMD V AFQVLAQLIKESL

CCLB 1: Kynurenine- MAKQLQARRLDGIDYNPWVEFVKLASEHDVVNLGQGFPDFP -oxoglutarate PPDFAVE AFQH A VS GDFMLNQYTKTFGYPPLTKILAS FFGELL transaminase 1 from GQEIDPLRNVLVTVGGYGALFTAFQALVDEGDEVIIIEPFFDC homo sapiens YEPMTMM AGGRP VFVS LKPGPIQNGELGS S S NWQLDPMEL A SEQ ID NO: 125 GKFTSRTKALVLNTPNNPLGKVFSREELELVASLCQQHDVVCI

TDE V YQWM V YD GHQHIS IAS LPGM WERTLTIGS AGKTFS ATG

WKVGWVLGPDHIMKHLRTVHQNSVFHCPTQSQAAVAESFER

EQLLFRQPS S YFVQFPQ AMQRCRDHMIRS LQS VGLKPIIPQGS

YFLITDISDFKRKMPDLPGAVDEPYDRRFVKWMIKNKGLVAI

PVSIFYSVPHQKHFDHYIRFCFVKDEATLQAMDEKLRKWKVE

L

CCLB2: kynurenine— MFL AQRS LCS LS GR AKFLKTIS S S KILGFS TS AKMS LKFTN AKR oxoglutarate IEGLDS N VWIEFT KL A ADPS V VNLGQGFPDIS PPT Y VKEELS KI transaminase 3 from AAIDSLNQYTRGFGHPSLVKALSYLYEKLYQKQIDSNKEILVT homo sapiens VGAYGSLFNTIQALIDEGDEVILIVPFYDCYEPMVRMAGATPV SEQ ID NO: 126 FIPLRS KPVYGKRWS S SDWTLDPQELES KFNS KTKAIILNTPHN

PLGKVYNREELQVIADLCIKYDTLCISDEVYEWLVYSGNKHL

KI ATFPGM WERTITIGS AGKTFS VTGWKLG WS IGPNHLIKHLQ

TVQQNTIYTCATPLQEALAQAFWIDIKRMDDPECYFNSLPKEL

EVKRDRMVRLLESVGLKPIVPDGGYFIIADVSLLDPDLSDMK

NNEPYD YKFVKWMTKHKKLS AIPVS AFCNS ETKS QFEKFVRF

CFIKKDSTLDAAEEIIKAWSVQKS

TnaA: tryptophanase MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE from E. coli DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE SEQ ID NO: 183 SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM

VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN

FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAM

YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE

TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT

LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA

QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFP

AQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRL

TIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRH

FTAKLKEV

[002] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence or nucleic acid sequence encoding a polypeptide of Table 29 or a functional fragment thereof. In some embodiments, the genetically engineered

bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 29 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 29 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 29 or a functional fragment thereof.

[003] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding tryptophan decarboxylase. In some embodiments, the Tryptophan Decarboxylase encoded by bacterium has at least about 80% identity with the entire sequence selected from SEQ ID NO: 97, 98, 99. In some embodiments, the Tryptophan Decarboxylase gene has at least about 85% identity with the entire sequence selected from SEQ ID NO: 97, 98, 99. In some embodiments, the

Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 90% identity with the entire sequence selected from SEQ ID NO: 97, 98, 99. In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 95% identity with the entire sequence selected from SEQ ID NO: 97, 98, 99. In another embodiment, the Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 96%, 97%, 98%, or 99% identity with the entire sequence selected from SEQ ID NO: 97, 98, 99. Accordingly, In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence selected from SEQ ID NO: 97, 98, 99. In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria comprises the sequence selected from SEQ ID NO: 97, 98, 99. In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria consists of the sequence of selected from SEQ ID NO: 97, 98, 99..

[0671] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding tryptophan aminotransferase. In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 80% identity with the entire sequence of selected form SEQ ID NO: 100 and 103. In another embodiment, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 100 and 103. In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 100 and 103. In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 100 and 103. In another embodiment, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100 and 103. Accordingly, In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100 and 103. In another embodiment, the Trp aminotransferase polypeptide encoded by the gene sequence comprises the sequence of SEQ ID NO: 100 and 103. In yet another embodiment the Trp aminotransferase polypeptide encoded by the gene sequence consists of the sequence of SEQ ID NO: 100 and 103.

[0672] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Monoamine oxidase (TYNA), e.g., from E. coli. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 101. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 101. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 101. Accordingly, In some embodiments, the gene sequence encodes a

polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 101. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 101. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 101.

[0673] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AAOl: Indole- 3 -acetaldehyde oxidase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 102. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 102. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 102. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 102. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 102. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 102.

In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding aspC: aspartate aminotransferase, e.g., from E. coli. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 104. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 104. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 104. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 104. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 104. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 104.

[0674] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TAA1: L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 105. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 105. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 105. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 105. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 105. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 105. [0675] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding STAO: L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 106. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 106. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 106. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 106. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 106. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 106.

[0676] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding ipdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae. In some

embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 107. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 107. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 107. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 107. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 107. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 107.

[0677] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding IAD1: Indole- 3 -acetaldehyde dehydrogenase, e.g., from Ustilago maydis. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 108. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 108. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 108. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 108. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 108. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 108.

[0678] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding YUC2: indole-3-pyruvate monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 109. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 109. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 109. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 109. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 109. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 109.

[0679] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding IaaM: Tryptophan 2-monooxygenase, e.g., from Pseudomonas savastanoi. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 110. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 110. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 110. Accordingly, in some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 110. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 110. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 110.

[0680] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding iaaH: Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 111. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 111. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 111. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 111. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 111. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 111.

[0681] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpDH: Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 112. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 112. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 112. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 112. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 112. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 112.

[0682] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CYP79B2: tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 113. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 113. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 113. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 113. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 113. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 113.

[0683] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CYP79B3: tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 114. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 114. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 114. Accordingly, in some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 114. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 114. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 114.

[0684] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CYP71A13: indoleacetaldoxime dehydratase, eg., from Arabidopis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 115. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 115. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 115. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 115. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 115. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 115.

[0685] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding PEN2: myrosinase, e.g, from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 116. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 116. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 116. Accordingly, In some embodiments, the gene sequence encodes a

polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 116. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 116. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 116.

[0686] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Nitl: Nitrilase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 117. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 117. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 117. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 117. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 117. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 117. [0687] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding IDOl: indoleamine 2,3-dioxygenase, e.g.,, from homo sapiens In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 118. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 118. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 118. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 118. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 118. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 118.

[0688] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TD02: tryptophan 2,3-dioxygenase, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 119. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 119. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 119. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 119. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 119. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 119.

[0689] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding BNA2: indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 120. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 120. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 120. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 120. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 120. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 120.

[0690] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Afmid: Kynurenine formamidase, e.g., from mouse. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 121. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 121. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 121. Accordingly, In some embodiments, the gene sequence encodes a

polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 121. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 121. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 121.

[0691] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding BNA3: kynurenine— oxoglutarate transaminase, e.g., from S. cerevisae. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 122. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 122. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 122. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 122. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 122. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 122. [0692] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding GOT2: Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 123. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 123. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 123. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 123. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 123. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID

NO: 123.

[0693] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AADAT: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 124. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 124. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 124. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 124. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 124. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 124.

[0694] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CCLB 1: Kynurenine— oxoglutarate transaminase 1, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 125. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 125. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 125. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 125. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 125. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 125.

[0695] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CCLB2: kynurenine— oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 126. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 126. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 126. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 126. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 126. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 126.

[0696] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TnaA: tryptophanase, e.g., from E. coli. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 183. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 183. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 183. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 183. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 183. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 183.

[0697] In some embodiments, TNA

(e.g., SEQ ID NO: 183) is mutated or deleted.

[0698]

[0699]

[0700] In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In som embodiments, the bacteria further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptamine exporter. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

[0701]

[0702] Table 16 depicts non-limiting examples of contemplated polypeptide sequences, which are encoded by indole-3- propionate producing bacteria.

Table 30. Non-limiting Examples of Sequences for indole-3-propionate Production

PSPFSGFDLFNHMAVAVCARGTQEAADAFKMLADEYEENVKT

SEQ ID NO: 128 GKSTYRGEEKQRILFEGIACWPYLRHKLTKLSEYGMNVTATV

YAEAFGVIYENMDELMAAYNKVPNSISFENALKMRLNAVTST

NTEGAVIHINRSCKLWSGFLYELARRLEKETGIPVVSFDGDQA

DPRNFSEAQYDTRIQGLNEVMVAKKEAE

FldC: subunit of MSNSDKFFNDFKDIVENPKKYIMKHMEQTGQKAIGCMPLYTP indole- 3 -lactate EELVLAAGMFPVGVWGSNTELSKAKTYFPAFICSILQTTLENA dehydratase from LNGEYDMLSGMMITNYCDSLKCMGQNFKLTVENIEFIPVTVPQ Clostridium NRKMEAGKEFLKSQYKMNIEQLEKISGNKITDESLEKAIEIYDE sporogenes HRKVMNDFSMLASKYPGIITPTKRNYVMKSAYYMDKKEHTE

KVRQLMDEIKAIEPKPFEGKRVITTGIIADSEDLLKILEENNIAIV

SEQ ID NO: 129 GDDIAHESRQYRTLTPEANTPMDRLAEQFANRECSTLYDPEKK

RGQYIVEMAKERKADGIIFFMTKFCDPEEYDYPQMKKDFEEA

GIPHVLIETDMQMKNYEQARTAIQAFSETL

FldD: indole-3- MFFTEQHELIRKLARDFAEQEIEPIADEVDKTAEFPKEIVKKMA acrylyl-CoA QNGFFGIKMPKE YGG AG ADNRAYVTIMEEISRAS GV AGIYLS S reductase from PNSLLGTPFLLVGTDEQKEKYLKPMIRGEKTLAFALTEPGAGS Clostridium DAGALATTAREEGDYYILNGRKTFITGAPISDNIIVFAKTDMSK sporogenes GTKGITTFIVDSKQEGVSFGKPEDKMGMIGCPTSDIILENVKVH

KSDILGEVNKGFITAMKTLSVGRIGVASQALGIAQAAVDEAVK

SEQ ID NO: 130 YAKQRKQFNRPIAKFQAIQFKLANMETKLNAAKLLVYNAAYK

MDCGEKADKEASMAKYFAAESAIQIVNDALQIHGGYGYIKDY

KIERLYRDVRVIAIYEGTSEVQQMVIASNLLK

FldHl: indole-3- MKILAYCVRPDEVDSFKKFSEKYGHTVDLIPDSFGPNVAHLAK lactate GYDGISILGNDTCNREALEKIKDCGIKYLATRTAGVNNIDFDA dehydrogenase AKEFGIN V AN VP A YS PNS VS EFTIGL ALS LTRKIPF ALKR VELN from Clostridium NFALGGLIGVELRNLTLGVIGTGRIGLKVIEGFSGFGMKKMIGY sporogenes DIFENEEAKKYIEYKSLDEVFKEADIITLHAPLTDDNYHMIGKE

SIAKMKDGVFIINAARGALIDSEALIEGLKSGKIAGAALDSYEY

SEQ ID NO: 131 EQGVFHNNKMNEIMQDDTLERLKSFPNVVITPHLGFYTDEAVS

NM VEITLMNLQEFELKGTC KNQRVC K

FldH2: indole-3- MKILMYSVREHEKPAIKKWLEANPGVQIDLCNNALSEDTVCK lactate AKEYDGIAIQQTNSIGGKAVYSTLKEYGIKQIASRTAGVDMIDL dehydrogenase KMASDSNILVTNVPAYSPNAIAELAVTHTMNLLRNIKTLNKRI from Clostridium AYGDYRWSADLIAREVRSVTVGVVGTGKIGRTSAKLFKGLGA sporogenes NVIGYDAYPDKKLEENNLLTYKESLEDLLREADVVTLHTPLLE

STKYMINKNNLKYMKPDAFIVNTGRGGIINTEDLIEALEQNKIA

SEQ ID NO: 132 GAALDTFENEGLFLNKVVDPTKLPDSQLDKLLKMDQVLITHH

VGFFTTTAVQNIVDTSLDSVVEVLKTNNSVNKVN

Acul: acrylyl- MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYK CoA reductase D ALAITGKAPVVRRFPMVPGIDFTGT VAQS S HADFKPGDRVIL from Rhodobacter NGWGVGEKHWGGLAERARVRGDWLVPLPAPLDLRQAAMIG sphaeroides TAGYTAMLCVLALERHGVVPGNGEIVVSGAAGGVGSVATTLL

AAKGYEVAAVTGRASEAEYLRGLGAASVIDRNELTGKVRPLG

SEQ ID NO: 133 QERWAGGIDVAGSTVLANMLSMMKYRGVVAACGLAAGMDL

PASVAPFILRGMTLAGVDSVMCPKTDRLAAWARLASDLDPAK LEEMTTELPFS E VIET APKFLD GT VRGRI VIP VTP [0703] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes. In some embodiments, FldA has at least about 80% identity with SEQ ID NO: 127. In some embodiments, FldA has at least about 85% identity with one or more of SEQ ID NO: 127. In some embodiments, FldA has at least about 90% identity with SEQ ID NO: 127. In some embodiments, FldA has at least about 95% identity with SEQ ID NO: 127. In some embodiments, FldA has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 127.

Accordingly, In some embodiments, FldA has at least about 80%, 81%, 82%, 83%, 127%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 127. In some embodiments,FldA comprises the sequence of SEQ ID NO: 127. In some embodiments, FldA consists of the sequence of one or more of SEQ ID NO: 84.

[0704] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldB: subunit of indole- 3 -lactate dehydratase, e.g., from Clostridium sporogenes. In some embodiments, FldB has at least about 80% identity with SEQ ID NO: 128. In some embodiments, FldB has at least about 85% identity with one or more of SEQ ID NO: 128. In some embodiments, FldB has at least about 90% identity with SEQ ID NO: 128. In some embodiments, FldB has at least about 95% identity with SEQ ID NO: 128. In some embodiments, FldB has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 128. Accordingly, In some embodiments, FldB has at least about 80%, 81%, 82%, 83%, 128%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 128. In some embodiments,FldB comprises the sequence of SEQ ID NO: 128. In some embodiments, FldB consists of the sequence of one or more of SEQ ID NO: 128.

[0705] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldC: subunit of indole- 3 -lactate dehydratase from Clostridium sporogenes. In some embodiments, FldC has at least about 80% identity with SEQ ID NO: 129. In some embodiments, FldC has at least about 85% identity with one or more of SEQ ID NO: 129. In some embodiments, FldC has at least about 90% identity with SEQ ID NO: 129. In some embodiments, FldC has at least about 95% identity with SEQ ID NO: 129. In some embodiments, FldC has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 129. Accordingly, In some embodiments, FldC has at least about 80%, 81%, 82%, 83%, 129%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 129. In some embodiments,FldC comprises the sequence of SEQ ID NO:

129. In some embodiments, FldC consists of the sequence of one or more of SEQ ID NO: 129.

[0706] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes. In some embodiments, FldD has at least about 80% identity with SEQ ID NO: 130. In some embodiments, FldD has at least about 85% identity with one or more of SEQ ID NO: 130. In some embodiments, FldD has at least about 90% identity with SEQ ID NO: 130. In some embodiments, FldD has at least about 95% identity with SEQ ID NO: 130. In some embodiments, FldD has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 130. Accordingly, In some embodiments, FldD has at least about 80%, 81%, 82%, 83%, 130%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 130. In some embodiments,FldD comprises the sequence of SEQ ID NO:

130. In some embodiments, FldD consists of the sequence of one or more of SEQ ID NO: 130.

[0707] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldHl: indole- 3 -lactate dehydrogenase, e.g., from Clostridium sporogenes. In some embodiments, FldHl has at least about 80% identity with SEQ ID NO: 131. In some embodiments, FldHl has at least about 85% identity with one or more of SEQ ID NO: 131. In some embodiments, FldHl has at least about 90% identity with SEQ ID NO: 131. In some embodiments, FldHl has at least about 95% identity with SEQ ID NO: 131. In some embodiments, FldHl has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 131. Accordingly, In some embodiments, FldHl has at least about 80%, 81%, 82%, 83%, 131%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 131. In some embodiments,FldHl comprises the sequence of SEQ ID NO: 131. In some embodiments, FldHl consists of the sequence of one or more of SEQ ID NO: 131. [0708] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldH2: indole- 3 -lactate dehydrogenase from

Clostridium sporogenes. In some embodiments, FldH2 has at least about 80% identity with SEQ ID NO: 132. In some embodiments, FldH2 has at least about 85% identity with one or more of SEQ ID NO: 132. In some embodiments, FldH2 has at least about 90% identity with SEQ ID NO: 132. In some embodiments, FldH2 has at least about 95% identity with SEQ ID NO: 132. In some embodiments, FldH2 has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 132. Accordingly, In some embodiments, FldH2 has at least about 80%, 81%, 82%, 83%, 132%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 132. In some embodiments,FldH2 comprises the sequence of SEQ ID NO: 132. In some embodiments, FldH2 consists of the sequence of one or more of SEQ ID NO: 132.

[0709] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Acul: acrylyl-CoA reductase from Rhodobacter sphaeroides. In some embodiments, Acul has at least about 80% identity with SEQ ID NO: 133. In some embodiments, Acul has at least about 85% identity with one or more of SEQ ID NO: 133. In some embodiments, Acul has at least about 90% identity with SEQ ID NO: 133. In some embodiments, Acul has at least about 95% identity with SEQ ID NO: 133. In some embodiments, Acul has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 133. Accordingly, In some embodiments, Acul has at least about 80%, 81%, 82%, 83%, 133%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 133. In some embodiments,AcuI comprises the sequence of SEQ ID NO: 133. In some embodiments, Acul consists of the sequence of one or more of SEQ ID NO: 133.

[0710] In some embodiments, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 127 through SEQ ID NO: 133. In another embodiment, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133. In some embodiments, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133. In some embodiments, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133. In another embodiment, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133. Accordingly, In some embodiments, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133. In another embodiment, the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding tryptophan pathway catabolic enzyme which comprises the sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133. In yet another embodiment the genetically engineered bactierum comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which consists of the sequence of one or more SEQ ID NO: 127 through SEQ ID NO: 133.

[0711] In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of one or more indole pathway metabolites described herein from tryptophan or a tryptophan metabolite. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the genetically engineered bacteria additionally produce tryptophan and/or chorismate through any of the pathways described herein, e.g. FIG. 39, FIG45A and FIG. 45B. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.

[01] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose or tetracycline. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g. , high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. In some embodiments, the tryptophan synthesis and/or tryptophan catabolism cassette(s) is under control of an inducible promoter. Exemplary inducible promoters which may control the expression of the al teast one sequence(s) include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.

[0712] Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g. , thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more exporters for exporting biological molecules or substrates, such any of the exporters described herein or otherwise known in the art, (6) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (7) combinations of one or more of such additional circuits.

Tryptophan Repressor (TrpR)

[0713] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, e.g., sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. Tryptophan and Tryptophan MetaboliteTransport

[0714] Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell.

[0715] The inner membrane protein

YddG of E. coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

[0716] In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.

[0717] In some embodiments, the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L- tryptophan in Escherichia coli. In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP.

[0718] In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transporte gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.

[0719] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g. , a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[0720] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g. , a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate

embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[0721] In some embodiments, the native transporter gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome. [0722] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[0723] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter.

Nucleic Acids

[0724] In some embodiments, the disclosure provides novel nucleic acids for producing or catabolizing tryptophan and/or one of its metabolites. In some

embodiments, such nucleic acids comprise gene sequence(s) expressed by the genetically engineered bacteria.

[0725] In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In one of the nucleic acid embodiments, described herein, the nucleic acid sequence encoding the tryptophan production enzyme(s) comprisesTrpE and/or TRPA, and/or TrpB, and/or TrpC and/or TrpD. Accordingly, in one embodiment, the nucleic acid sequence comprising TrpE and/or TRPA, and/or TrpB, and/or TrpC and/or TrpD gene has at least about 80% identity with one or more of SEQ ID NO: 74, 76, 78, 80, 82, 84. In one embodiment, the nucleic acid sequence comprising TrpE and/or TRPA, and/or TrpB, and/or TrpC and/or TrpD gene has at least about 90% identity with SEQ ID NO: 26. In another embodiment, the nucleic acid sequence comprising TrpE and/or TrpA, and/or TrpB, and/or TrpC and/or TrpD gene has at least about 95% identity with one or more of SEQ ID NO: 74, 76, 78, 80, 82, 84. Accordingly, in one embodiment, the nucleic acid sequence comprising the TrpE and/or TRPA, and/or TrpB, and/or TrpC and/or TrpD gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 74, 76, 78, 80, 82, 84. In another embodiment, the nucleic acid sequence comprising the comprisesTrpE and/or TRPA, and/or TrpB, and/or TrpC and/or TrpD gene comprises one or more of SEQ ID NO: 74, 76, 78, 80, 82, 84. In yet another embodiment the nucleic acid sequence comprising the comprisesTrpE and/or TRPA, and/or TrpB, and/or TrpC and/or TrpD gene consists of one or more of SEQ ID NO: 74, 76, 78, 80, 82, 84.

[0726] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an anthranilate synthase component I (TrpE). In some embodiments, the nucleic acid comprises gene sequence encoding TrpE. In some embodiments, the nucleic acid comprises a TrpE gene sequence. In certain embodiments, the nucleic acid comprising the TrpE gene sequence has at least about 80% identity with SEQ ID NO: 74. In certain embodiments, the nucleic acid comprising the TrpE gene sequence has at least about 90% identity with SEQ ID NO: 74. In certain embodiments, the nucleic acid comprising the TrpE gene sequence has at least about 95% identity with SEQ ID NO: 74. In some embodiments, the nucleic acid comprising the TrpE gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 74. In some specific embodiments, the nucleic acid comprising the TrpE gene sequence comprises SEQ ID NO: 74. In other specific embodiments, the nucleic acid comprising the TrpE gene sequence consists of SEQ ID NO: 74. [0727] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an anthranilate synthase component II (TrpD). In some embodiments, the nucleic acid comprises gene sequence encoding TrpD. In some embodiments, the nucleic acid comprises a TrpD gene sequence. In certain embodiments, the nucleic acid comprising the TrpD gene sequence has at least about 80% identity with SEQ ID NO: 76. In certain embodiments, the nucleic acid comprising the TrpD gene sequence has at least about 90% identity with SEQ ID NO: 76. In certain embodiments, the nucleic acid comprising the TrpD gene sequence has at least about 95% identity with SEQ ID NO: 76. In some embodiments, the nucleic acid comprising the TrpD gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 76. In some specific embodiments, the nucleic acid comprising the TrpD gene sequence comprises SEQ ID NO: 76. In other specific embodiments, the nucleic acid comprising the TrpD gene sequence consists of SEQ ID NO: 76.

[0728] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an indole-3-glycerol phosphate synthase / phosphoribosylanthranilate isomerase (TrpC). In some embodiments, the nucleic acid comprises gene sequence encoding TrpC. In some embodiments, the nucleic acid comprises a TrpC gene sequence. In certain embodiments, the nucleic acid comprising the TrpC gene sequence has at least about 80% identity with SEQ ID NO: 78. In certain embodiments, the nucleic acid comprising the TrpC gene sequence has at least about 90% identity with SEQ ID NO: 78. In certain embodiments, the nucleic acid comprising the TrpC gene sequence has at least about 95% identity with SEQ ID NO: 78. In some embodiments, the nucleic acid comprising the TrpC gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 78. In some specific embodiments, the nucleic acid comprising the TrpC gene sequence comprises SEQ ID NO: 78. In other specific embodiments, the nucleic acid comprising the TrpC gene sequence consists of SEQ ID NO: 78. [0729] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an tryptophan synthase, β subunit dimer (TrpB). In some embodiments, the nucleic acid comprises gene sequence encoding TrpB. In some embodiments, the nucleic acid comprises a TrpB gene sequence. In certain embodiments, the nucleic acid comprising the TrpB gene sequence has at least about 80% identity with SEQ ID NO: 80. In certain embodiments, the nucleic acid comprising the TrpB gene sequence has at least about 90% identity with SEQ ID NO: 80. In certain embodiments, the nucleic acid comprising the TrpB gene sequence has at least about 95% identity with SEQ ID NO: 80. In some embodiments, the nucleic acid comprising the TrpB gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 80. In some specific embodiments, the nucleic acid comprising the TrpB gene sequence comprises SEQ ID NO: 80. In other specific embodiments, the nucleic acid comprising the TrpB gene sequence consists of SEQ ID NO: 80.

[0730] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding a tryptophan synthase, a subunit (TrpA). In some embodiments, the nucleic acid comprises gene sequence encoding TrpA. In some embodiments, the nucleic acid comprises a TrpA gene sequence. In certain embodiments, the nucleic acid comprising the TrpA gene sequence has at least about 80% identity with SEQ ID NO: 82. In certain embodiments, the nucleic acid comprising the TrpA gene sequence has at least about 90% identity with SEQ ID NO: 82. In certain embodiments, the nucleic acid comprising the TrpA gene sequence has at least about 95% identity with SEQ ID NO: 82. In some embodiments, the nucleic acid comprising the TrpA gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 82. In some specific embodiments, the nucleic acid comprising the TrpA gene sequence comprises SEQ ID NO: 82. In other specific embodiments, the nucleic acid comprising the TrpA gene sequence consists of SEQ ID NO: 82. [0731] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding feedback resistant TrpE (TrpEfbr). In some embodiments, the nucleic acid comprises gene sequence encoding TrpEfbr. In some embodiments, the nucleic acid comprises a TrpEfbr gene sequence. In certain embodiments, the nucleic acid comprising the TrpEfbr gene sequence has at least about 80% identity with SEQ ID NO: 185. In certain embodiments, the nucleic acid comprising the TrpEfbr gene sequence has at least about 90% identity with SEQ ID NO: 185. In certain embodiments, the nucleic acid comprising the TrpEfbr gene sequence has at least about 95% identity with SEQ ID NO: 185. In some embodiments, the nucleic acid comprising the TrpEfbr gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 185. In some specific embodiments, the nucleic acid comprising the TrpEfbr gene sequence comprises SEQ ID NO: 185. In other specific embodiments, the nucleic acid comprising the TrpEfbr gene sequence consists of SEQ ID NO: 185.

[0732] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding feedback resistant 2-dehydro-3- deoxyphosphoheptonate aldolase (AroGfbr). In some embodiments, the nucleic acid comprises gene sequence encoding AroGfbr. In some embodiments, the nucleic acid comprises a AroGfbr gene sequence. In certain embodiments, the nucleic acid comprising the AroGfbr gene sequence has at least about 80% identity with SEQ ID NO: 167. In certain embodiments, the nucleic acid comprising the AroGfbr gene sequence has at least about 90% identity with SEQ ID NO: 167. In certain

embodiments, the nucleic acid comprising the AroGfbr gene sequence has at least about 95% identity with SEQ ID NO: 167. In some embodiments, the nucleic acid comprising the AroGfbr gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 167. In some specific embodiments, the nucleic acid comprising the AroGfbr gene sequence comprises SEQ ID NO: 167. In other specific embodiments, the nucleic acid

comprising the AroGfbr gene sequence consists of SEQ ID NO: 167. [0733] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding 2-oxoglutarate reductase ((S)-2- hydroxyglutarate-forming) (SerAfbr). In some embodiments, the nucleic acid comprises gene sequence encoding SerAfbr. In some embodiments, the nucleic acid comprises a SerAfbr gene sequence. In certain embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 80% identity with SEQ ID NO: 169. In certain embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 90% identity with SEQ ID NO: 169. In certain embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 95% identity with SEQ ID NO: 169. In some embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169. In some specific embodiments, the nucleic acid comprising the SerAfbr gene sequence comprises SEQ ID NO: 169. In other specific embodiments, the nucleic acid comprising the SerAfbr gene sequence consists of SEQ ID NO: 169.

[0734] In some embodiments, the disclosure provides novel nucleic acids for producing tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding one or more tryptophan production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding feedback resistant 2-oxoglutarate reductase ((S)-2-hydroxyglutarate-forming) (SerAfbr). In some embodiments, the nucleic acid comprises gene sequence encoding SerAfbr. In some embodiments, the nucleic acid comprises a SerAfbr gene sequence. In certain embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 80% identity with SEQ ID NO: 179. In certain embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 90% identity with SEQ ID NO: 179. In certain

embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 95% identity with SEQ ID NO: 179. In some embodiments, the nucleic acid comprising the SerAfbr gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 179. In some specific embodiments, the nucleic acid comprising the SerAfbr gene sequence comprises SEQ ID NO: 179. In other specific embodiments, the nucleic acid comprising the SerAfbr gene sequence consists of SEQ ID NO: 179.

[0735] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding kynureninase, e.g., from Pseudomonas fluorescens. In some embodiments, the nucleic acid comprises gene sequence encoding KynU. In some embodiments, the nucleic acid comprises a KynU gene sequence. In certain

embodiments, the nucleic acid comprising the KynU gene sequence has at least about 80% identity with SEQ ID NO: 92. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 90% identity with SEQ ID NO: 92. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 95% identity with SEQ ID NO: 92. In some embodiments, the nucleic acid comprising the KynU gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 92. In some specific embodiments, the nucleic acid comprising the KynU gene sequence comprises SEQ ID NO: 92. In other specific embodiments, the nucleic acid comprising the KynU gene sequence consists of SEQ ID NO: 92.

[0736] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding kynureninase, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding KynU. In some embodiments, the nucleic acid comprises a KynU gene sequence. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 80% identity with SEQ ID NO: 93. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 90% identity with SEQ ID NO: 93. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 95% identity with SEQ ID NO: 93. In some embodiments, the nucleic acid comprising the KynU gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 93. In some specific embodiments, the nucleic acid comprising the KynU gene sequence comprises SEQ ID NO: 93. In other specific embodiments, the nucleic acid comprising the KynU gene sequence consists of SEQ ID NO: 93.

[0737] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding kynureninase, e.g., from Shewanella. In some embodiments, the nucleic acid comprises gene sequence encoding KynU. In some embodiments, the nucleic acid comprises a KynU gene sequence. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 80% identity with SEQ ID NO: 94. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 90% identity with SEQ ID NO: 94. In certain embodiments, the nucleic acid comprising the KynU gene sequence has at least about 95% identity with SEQ ID NO: 94. In some embodiments, the nucleic acid comprising the KynU gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 94. In some specific embodiments, the nucleic acid comprising the KynU gene sequence comprises SEQ ID NO: 94. In other specific embodiments, the nucleic acid comprising the KynU gene sequence consists of SEQ ID NO: 94.

[0738] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Trp aminotransferase (EC 2.6.1.27), e.g., tryptophan

aminotransferase from Cryptococcus deuterogattii R265. In some embodiments, the nucleic acid comprises gene sequence encoding Trp aminotransferase. In some embodiments, the nucleic acid comprises a Trp aminotransferase gene sequence. In certain embodiments, the nucleic acid comprising the Trp aminotransferase gene sequence has at least about 80% identity with SEQ ID NO: 95. In certain embodiments, the nucleic acid comprising the Trp aminotransferase gene sequence has at least about 90% identity with SEQ ID NO: 95. In certain embodiments, the nucleic acid comprising the Trp aminotransferase gene sequence has at least about 95% identity with SEQ ID NO: 95. In some embodiments, the nucleic acid comprising the Trp aminotransferase gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95. In some specific embodiments, the nucleic acid comprising the Trp aminotransferase gene sequence comprises SEQ ID NO: 95. In other specific embodiments, the nucleic acid comprising the Trp aminotransferase gene sequence consists of SEQ ID NO: 95.

[0739] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Tryptophan Decarboxylase (EC 4.1.1.28) Chain A, e.g., from Ruminococcus Gnavus. In some embodiments, the nucleic acid comprises gene sequence encoding Tdc. In some embodiments, the nucleic acid comprises a Tdc gene sequence. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 80% identity with SEQ ID NO: 96. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 90% identity with SEQ ID NO: 96. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 96% identity with SEQ ID NO: 96. In some embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96. In some specific embodiments, the nucleic acid comprising the Tdc gene sequence comprises SEQ ID NO: 96. In other specific embodiments, the nucleic acid comprising the Tdc gene sequence consists of SEQ ID NO: 96.

[0740] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Tryptophan Decarboxylase e.g., from C. roseus. In some embodiments, the nucleic acid comprises gene sequence encoding Tdc. In some embodiments, the nucleic acid comprises a Tdc gene sequence. In certain

embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 80% identity with SEQ ID NO: 171. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 90% identity with SEQ ID NO: 171. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 171% identity with SEQ ID NO: 171. In some embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 171. In some specific embodiments, the nucleic acid comprising the Tdc gene sequence comprises SEQ ID NO: 171. In other specific embodiments, the nucleic acid comprising the Tdc gene sequence consists of SEQ ID NO: 171.

[0741] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Tryptophan Decarboxylase e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding Tdc . In some embodiments, the nucleic acid comprises a Tdc gene sequence. In certain

embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 80% identity with SEQ ID NO: 173. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 90% identity with SEQ ID NO: 173. In certain embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 173% identity with SEQ ID NO: 173. In some embodiments, the nucleic acid comprising the Tdc gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 173. In some specific embodiments, the nucleic acid comprising the Tdc gene sequence comprises SEQ ID NO: 173. In other specific embodiments, the nucleic acid comprising the Tdc gene sequence consists of SEQ ID NO: 173.

[0742] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding tryptophan dehydrogenase (TrpDH). In some embodiments, the nucleic acid comprises gene sequence encoding TrpDH . In some embodiments, the nucleic acid comprises a TrpDH gene sequence. In certain embodiments, the nucleic acid comprising the TrpDH gene sequence has at least about 80% identity with SEQ ID NO: 175. In certain embodiments, the nucleic acid comprising the TrpDH gene sequence has at least about 90% identity with SEQ ID NO: 175. In certain

embodiments, the nucleic acid comprising the TrpDH gene sequence has at least about 175% identity with SEQ ID NO: 175. In some embodiments, the nucleic acid comprising the TrpDH gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

175. In some specific embodiments, the nucleic acid comprising the TrpDH gene sequence comprises SEQ ID NO: 175. In other specific embodiments, the nucleic acid comprising the TrpDH gene sequence consists of SEQ ID NO: 175.

[0743] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding indolepyruvate/phenylpyruvate decarboxylase (IpdC). In some embodiments, the nucleic acid comprises gene sequence encoding IpdC. In some embodiments, the nucleic acid comprises a IpdC gene sequence. In certain

embodiments, the nucleic acid comprising the IpdC gene sequence has at least about 80% identity with SEQ ID NO: 176. In certain embodiments, the nucleic acid comprising the IpdC gene sequence has at least about 90% identity with SEQ ID NO:

176. In certain embodiments, the nucleic acid comprising the IpdC gene sequence has at least about 176% identity with SEQ ID NO: 176. In some embodiments, the nucleic acid comprising the IpdC gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 176. In some specific embodiments, the nucleic acid comprising the IpdC gene sequence comprises SEQ ID NO: 176. In other specific embodiments, the nucleic acid comprising the IpdC gene sequence consists of SEQ ID NO: 176.

[0744] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding ladl. In some embodiments, the nucleic acid comprises gene sequence encoding ladl . In some embodiments, the nucleic acid comprises a ladl gene sequence. In certain embodiments, the nucleic acid comprising the ladl gene sequence has at least about 80% identity with SEQ ID NO: 177. In certain embodiments, the nucleic acid comprising the Iadl gene sequence has at least about 90% identity with SEQ ID NO: 177. In certain embodiments, the nucleic acid comprising the Iadl gene sequence has at least about 177% identity with SEQ ID NO: 177. In some

embodiments, the nucleic acid comprising the Iadl gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 177. In some specific embodiments, the nucleic acid comprising the Iadl gene sequence comprises SEQ ID NO: 177. In other specific embodiments, the nucleic acid comprising the Iadl gene sequence consists of SEQ ID NO: 177.

[0745] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Flavodoxin 1 (FldA). In some embodiments, the nucleic acid comprises gene sequence encoding FldA. In some embodiments, the nucleic acid comprises a FldA gene sequence. In certain embodiments, the nucleic acid comprising the FldA gene sequence has at least about 80% identity with SEQ ID NO: 187. In certain embodiments, the nucleic acid comprising the FldA gene sequence has at least about 90% identity with SEQ ID NO: 187. In certain embodiments, the nucleic acid comprising the FldA gene sequence has at least about 187% identity with SEQ ID NO: 187. In some embodiments, the nucleic acid comprising the FldA gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 187. In some specific embodiments, the nucleic acid comprising the FldA gene sequence comprises SEQ ID NO: 187. In other specific embodiments, the nucleic acid comprising the FldA gene sequence consists of SEQ ID NO: 187.

[0746] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Flavodoxin 2 (FldB). In some embodiments, the nucleic acid comprises gene sequence encoding FldB. In some embodiments, the nucleic acid comprises a FldB gene sequence. In certain embodiments, the nucleic acid comprising the FldB gene sequence has at least about 80% identity with SEQ ID NO: 188. In certain embodiments, the nucleic acid comprising the FldB gene sequence has at least about 90% identity with SEQ ID NO: 188. In certain embodiments, the nucleic acid comprising the FldB gene sequence has at least about 188% identity with SEQ ID NO: 188. In some embodiments, the nucleic acid comprising the FldB gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 188. In some specific embodiments, the nucleic acid comprising the FldB gene sequence comprises SEQ ID NO: 188. In other specific embodiments, the nucleic acid comprising the FldB gene sequence consists of SEQ ID NO: 188.

[0747] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding FldC. In some embodiments, the nucleic acid comprises gene sequence encoding FldC. In some embodiments, the nucleic acid comprises a FldC gene sequence. In certain embodiments, the nucleic acid comprising the FldC gene sequence has at least about 80% identity with SEQ ID NO: 189. In certain embodiments, the nucleic acid comprising the FldC gene sequence has at least about 90% identity with SEQ ID NO: 189. In certain embodiments, the nucleic acid comprising the FldC gene sequence has at least about 189% identity with SEQ ID NO: 189. In some

embodiments, the nucleic acid comprising the FldC gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 189. In some specific embodiments, the nucleic acid comprising the FldC gene sequence comprises SEQ ID NO: 189. In other specific embodiments, the nucleic acid comprising the FldC gene sequence consists of SEQ ID NO: 189.

[0748] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding Acul. In some embodiments, the nucleic acid comprises gene sequence encoding Acul. In some embodiments, the nucleic acid comprises a Acul gene sequence. In certain embodiments, the nucleic acid comprising the Acul gene sequence has at least about 80% identity with SEQ ID NO: 190. In certain embodiments, the nucleic acid comprising the Acul gene sequence has at least about 90% identity with SEQ ID NO: 190. In certain embodiments, the nucleic acid comprising the Acul gene sequence has at least about 190% identity with SEQ ID NO: 190. In some

embodiments, the nucleic acid comprising the Acul gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 190. In some specific embodiments, the nucleic acid comprising the Acul gene sequence comprises SEQ ID NO: 190. In other specific embodiments, the nucleic acid comprising the Acul gene sequence consists of SEQ ID NO: 190.

[0749] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding FldHl. In some embodiments, the nucleic acid comprises gene sequence encoding FldHl. In some embodiments, the nucleic acid comprises a FldHl gene sequence. In certain embodiments, the nucleic acid comprising the FldHl gene sequence has at least about 80% identity with SEQ ID NO: 191. In certain

embodiments, the nucleic acid comprising the FldHl gene sequence has at least about 90% identity with SEQ ID NO: 191. In certain embodiments, the nucleic acid comprising the FldHl gene sequence has at least about 191% identity with SEQ ID NO: 191. In some embodiments, the nucleic acid comprising the FldHl gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 191. In some specific embodiments, the nucleic acid comprising the FldHl gene sequence comprises SEQ ID NO: 191. In other specific embodiments, the nucleic acid comprising the FldHl gene sequence consists of SEQ ID NO: 191.

[0750] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more enzyme(s) for the production of one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding FldD. In some embodiments, the nucleic acid comprises gene sequence encoding FldD. In some embodiments, the nucleic acid comprises a FldD gene sequence. In certain embodiments, the nucleic acid comprising the FldD gene sequence has at least about 80% identity with SEQ ID NO: 193. In certain embodiments, the nucleic acid comprising the FldD gene sequence has at least about 90% identity with SEQ ID NO: 193. In certain embodiments, the nucleic acid comprising the FldD gene sequence has at least about 193% identity with SEQ ID NO: 193. In some

embodiments, the nucleic acid comprising the FldD gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 193. In some specific embodiments, the nucleic acid comprising the FldD gene sequence comprises SEQ ID NO: 193. In other specific embodiments, the nucleic acid comprising the FldD gene sequence consists of SEQ ID NO: 193.

[0751] In some embodiments, the nucleic acid comprises gene sequence encoding TrpE. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 75. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 75. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 75. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 75. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 75. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 75.

[0752] In some embodiments, the nucleic acid comprises gene sequence encoding TrpA. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 83. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 83. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 83. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 83. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 83. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 83.

[0753] In some embodiments, the nucleic acid comprises gene sequence encoding TrpB. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 81. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 81. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 81. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 81. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 81. In other embodiments, the nucleic acid In some embodiments, the nucleic acid comprises gene sequence encoding TrpD. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 77. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 77. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 77. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 77. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 77. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 77.

[0754] In some embodiments, the nucleic acid comprises gene sequence encoding TrpC. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 79. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 79. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 79. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 79. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 79.

[0755] In some embodiments, the nucleic acid comprises gene sequence encoding AroGfbr: feedback resistant 2-dehydro-3-deoxyphosphoheptonate aldolase, e.g., from E. coli. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 84. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 84. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 84. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

84. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 84. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 84.

[0756] In some embodiments, the nucleic acid comprises gene sequence encoding TrpEfbr: feedback resistant anthranilate synthase component I, e.g., from E. coli. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 85. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 85. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 85. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

85. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 85. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 85

[0757] In some embodiments, the nucleic acid comprises gene sequence encoding SerA: 2-oxoglutarate reductase, e.g., from E. coli Nissle. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 86. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 86. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

86. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 86. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 86. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 86.

[0758] In some embodiments, the nucleic acid comprises gene sequence encoding SerAfbr: feedback resistant 2-oxoglutarate reductase, e.g., from E. coli Nissle. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 87. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 87. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 87. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

87. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 87. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 87.

[0759] In some embodiments, the nucleic acid comprises gene sequence encoding TnaA: tryptophanase, e.g., from E. coli. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 88. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO:

88. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 88. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 88. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 88. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 88.

[0760] In some embodiments, the nucleic acid comprises gene sequence encoding TDC: Tryptophan decarboxylase, e.g., from Catharanthus roseus. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 97. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 97. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

97. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 97%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 97. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 97. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 97.

[0761] In some embodiments, the nucleic acid comprises gene sequence encoding TDC: Tryptophan decarboxylase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 98. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 98. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

98. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 98%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 98. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 98. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 98.

[0762] In some embodiments, the nucleic acid comprises gene sequence encoding Tryptophan Decarboxylase (EC 4.1.1.28) Chain A, , e.g., from Ruminococcus Gnavus Tryptophan. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 99. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 99. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 99. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 99%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

99. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 99. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 99.

[0763] In some embodiments, the nucleic acid comprises gene sequence encoding tryptophan aminotransferase, e.g., from Cryptococcus deuterogattii R265. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 100. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 100. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

100. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 100%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 100. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 100. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 100.

[0764] In some embodiments, the nucleic acid comprises gene sequence encoding AR09: L-tryptophan aminotransferase, e.g., from S. cerevisae. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 103. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 103. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 103. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 103%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 103. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 103. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 103.

[0765] In some embodiments, the nucleic acid comprises gene sequence encoding TYNA: Monoamine oxidase, e.g., from E. coli. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 101. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 101. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 101. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 101%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 101. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

101. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 101.

[0766] In some embodiments, the nucleic acid comprises gene sequence encoding AAOl: Indole- 3 -acetaldehyde oxidase, e.g., from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 102. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 102. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

102. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 102%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 102. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 102. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 102.

[0767] In some embodiments, the nucleic acid comprises gene sequence encoding aspC: aspartate aminotransferase, e.g., from E. coli. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 104. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 104. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 104. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 104%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 104. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

104. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 104.

[0768] In some embodiments, the nucleic acid comprises gene sequence encoding TAA1: L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 105. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 105. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 105. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 105%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

105. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 105. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 105.

[0769] In some embodiments, the nucleic acid comprises gene sequence encoding STAO: L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 106. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 106. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

106. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 106%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 106. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 106. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 106.

[0770] In some embodiments, the nucleic acid comprises gene sequence ipdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae. In some

embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 107. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 107. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

107. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 107%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 107. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 107. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 107.

[0771] In some embodiments, the nucleic acid comprises gene sequence IAD1: Indole- 3 -acetaldehyde dehydrogenase from Ustilago maydis. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 108. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 108. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 108. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 108%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 108. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

108. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 108.

[0772] In some embodiments, the nucleic acid comprises gene sequence YUC2: indole-3-pyruvate monoxygenase from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 109. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 109. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 109. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 109%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 109. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

109. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 109.

[0773] In some embodiments, the nucleic acid comprises gene sequence IaaM: Tryptophan 2-monooxygenase, e.g., from Pseudomonas savastanoi. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 110. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 110. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

110. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 110%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 110. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 110. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 110.

[0774] In some embodiments, the nucleic acid comprises gene sequence iaaH: Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 111. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 111. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 111. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 111%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 111. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 111. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 111.

[0775] In some embodiments, the nucleic acid comprises gene sequence TrpDH: Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 112. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 112. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

112. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 112%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 112. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 112. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 112.

[0776] In some embodiments, the nucleic acid comprises gene sequence

CYP79B2: tryptophan N-monooxygenase from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 113. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 113. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

113. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 113%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 113. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 113. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 113.

[0777] In some embodiments, the nucleic acid comprises gene sequence

CYP79B3: tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 114. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 114. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

114. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 114%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 114. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 114. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 114.

[0778] In some embodiments, the nucleic acid comprises gene sequence CYP71A13: indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 115. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 115. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

115. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 115%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 115. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 115. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 115.

[0779] In some embodiments, the nucleic acid comprises gene sequence PEN2: myrosinase, e.g., from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 116. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO:

116. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 116. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 116%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 116. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 116. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 116.

[0780] In some embodiments, the nucleic acid comprises gene sequence Nitl: Nitrilase, e.g., from Arabidopsis thaliana. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 117. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO:

117. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 117. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 117%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 117. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

117. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 117.

[0781] In some embodiments, the nucleic acid comprises gene sequence IDOl: indoleamine 2,3-dioxygenase, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 118. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO:

118. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 118. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 118%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 118. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 118. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 118.

[0782] In some embodiments, the nucleic acid comprises gene sequence TD02: tryptophan 2,3-dioxygenase, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 119. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 119. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 119. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 119%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 119. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

119. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 119.

[0783] In some embodiments, the nucleic acid comprises gene sequence BNA2: indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 120. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO:

120. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 120. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 120%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 120. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

120. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 120.

[0784] In some embodiments, the nucleic acid comprises gene sequence Afmid: Kynurenine formamidase, e.g., from mouse. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 121. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO:

121. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 121. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 121%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 121. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 121. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 121.

[0785] In some embodiments, the nucleic acid comprises gene sequence BNA3: kynurenine— oxoglutarate transaminase, e.g., from S. cerevisae. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 122. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 122. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 122. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 122%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 122. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO:

122. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 122.

[0786] In some embodiments, the nucleic acid comprises gene sequence GOT2: Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 123. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 123. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

123. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 123%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 123. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 123. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 123.

[0787] In some embodiments, the nucleic acid comprises gene sequence AADAT: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 124. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 124. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 124. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 124%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

124. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 124. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 124.

[0788] In some embodiments, the nucleic acid comprises gene sequence CCLB 1: Kynurenine— oxoglutarate transaminase 1, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 125. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 125. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

125. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 125%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 125. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 125. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 125.

[0789] In some embodiments, the nucleic acid comprises gene sequence CCLB2: kynurenine— oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 126. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 126. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

126. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 126%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 126. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 126. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 126.

[0790] In some embodiments, the nucleic acid comprises gene sequence TnaA: tryptophanase, e.g., from E. coli. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 183. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 183. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 183. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 183%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 183. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 183. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 183.

[0791] In some embodiments, the nucleic acid comprises gene sequence FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 127. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 127. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 127. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 127%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 127. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 127. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 127.

[0792] In some embodiments, the nucleic acid comprises gene sequence FldB: subunit of indole- 3 -lactate dehydratase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 128. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 128. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

128. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 128%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 128. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 128. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 128.

[0793] In some embodiments, the nucleic acid comprises gene sequence FldC: subunit of indole- 3 -lactate dehydratase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 129. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 129. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

129. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 129%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 129. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 129. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 129.

[0794] In some embodiments, the nucleic acid comprises gene sequence FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 130. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 130. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

130. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 130%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 130. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 130. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 130.

[0795] In some embodiments, the nucleic acid comprises gene sequence FldHl: indole- 3 -lactate dehydrogenase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 131. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 131. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

131. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 131%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 131. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 131. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 131.

[0796] In some embodiments, the nucleic acid comprises gene sequence FldH2: indole- 3 -lactate dehydrogenase, e.g., from Clostridium sporogenes. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 132. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 132. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO:

132. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 132%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 132. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 132. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 132.

[0797] In some embodiments, the nucleic acid comprises gene sequence Acul: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides . In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 133. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 133. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 133. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 133%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 133. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 133. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 133.

[0798] In any of the nucleic acid embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which function in production or catabolism of tryptophan or one or more of its metabolites is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0799] In any of the nucleic acid embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which function in production or catabolism of tryptophan or one or more of its metabolites is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., such as those found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20.

[0800] In any of the nucleic acid embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which function in production or catabolism of tryptophan or one or more of its metabolites is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21. In any of the nucleic acid

embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which function in production or catabolism of tryptophan or one or more of its metabolites is operably linked to a sequence that targets the gene sequence or its resultant polypeptide to a particular cellular location, including but not limited to a signal sequence, a secretion sequence, or an display anchor sequence.

[0801] In any of the nucleic acid embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which function in production or catabolism of tryptophan or one or more of its metabolites is modified and/or mutated (for example, by deletion, insertion, and/or substitution of

nucleotide(s))e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0802] In any of the nucleic acid embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which functions in production or catabolism of tryptophan or one or more of its metabolites may be codon optimized, e.g., to improve expression in the host microorganism.

[0803] In any of the nucleic acid embodiments, described above and elsewhere herein, the gene sequence encoding one or more polypeptides which functions in production or catabolism of tryptophan or one or more of its metabolites are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.

Diseases

In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of tryptophan and one or more of its metabolites described herein in the subject, (e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissue) for the treatment, management or prevention of any of the diseases, disorders, symptoms, or conditions described herein. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein (e.g., in FIG. 2) in the subject, e.g., e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissues. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein, in the subject, e.g., in the serum and/or in the or in the gut/and or in the nervous system and/or in the brain. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of tryptamine. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of indole-3-acetic acid. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of indole-3-propionic acid.

[0804] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the ratios of tryptophan and one or more of its metabolites relative to another tryptophan metabolite described herein in the subject, (e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissue) for the treatment, management or prevention of any of the diseases, disorders, symptoms, or conditions described herein. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues, e.g., for the treatment, prevention and/or management of one more of the diseases, disorders and conditions described below herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and

Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein.

[0805] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of tryptophan and one or more of its metabolites described herein in the subject, (e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissue) for the treatment, management or prevention of any of the diseases, disorders, symptoms, or conditions described herein. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues, e.g., for the treatment, prevention and/or management of one or more of the diseases, conditions, and/or disorders described herein. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the subject, e.g., in the serum and/or in the gut and/or in the brain or nervous system. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites, e.g., described in FIG. 2 and elsewhere herein, described herein in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein., in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of tryptamine. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of indole- 3 -acetic acid. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of indole-3-propionic acid.

[0806] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the ratios of tryptophan or one or more of its metabolites relative to another tryptophan metabolite described herein in the subject, (e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissue) for the treatment, management or prevention of any of the diseases, disorders, symptoms, or conditions described herein. In certain

embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues, e.g., for the treatment, prevention and/or management of one or more of the diseases, conditions, and/or disorders described herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein.

[0807] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of tryptophan and one or more of its metabolites described herein in the subjec, (e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissue) for the treatment, management or prevention of any of the diseases, disorders, symptoms, or conditions described herein. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues, e.g., for the treatment, prevention and/or management of one or more of the diseases, conditions, and/or disorders described herein. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites (e.g., as listed in FIG. 2) described herein in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein, in the subject, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of tryptamine. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of indole- 3 -acetic acid. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of indole-3-propionic acid.

[0808] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease ratios of tryptophan or one or more of its metabolites to another tryptophan metabolite described herein in the subject, (e.g., in the gut, the central nervous system, in the plasma, serum, blood, brain, a tumor, or other tissue) for the treatment, management or prevention of any of the diseases, disorders, symptoms, or conditions described herein. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the subject, e.g., e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues, e.g., for the treatment, prevention and/or management of one or more of the diseases, conditions, and/or disorders described herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in FIG. FIG. 2, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels, e.g., in the gut, the central nervous system, in the plasma, blood, brain, a tumor, or other tissues, for the prevention, treatment, or management of any of the diseases, disorders, conditions, or symtoms described herein. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of one or more of the diseases, disorders and conditions described herein. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

Immune function

[0809] In the immune system, tryptophan has been implicated in the immune regulation. The decreased concentration of this amino acids leads to cell cycle arrest and apoptosis. Trp catabolites are well known for their immunosuppressive functions, disease tolerance, and contribution to immune privileged sites such as eyes, brain, placenta, and testes. The KP represents >95% of Trp-catabolizing pathways and is now established as a key regulator of innate and adaptive immunity through its involvement in cancer, autoimmunity, and infection.

Cancer and Immune Evasion

[0810] T regulatory cells, or Tregs, are a subpopulation of T cells that modulate the immune system by preventing excessive immune reactions, maintaining tolerance to self- antigens, and abrogating autoimmunity. Tregs suppress the immune responses of other cells, for example, shutting down immune responses after they have successfully eliminated invading organisms. These cells generally suppress or downregulate induction and proliferation of effector T cells. Tregs have been found to be up- regulated in individuals with cancer and are often recruited to the sites of many tumors. Studies in both humans and animal models suggest that high levels of Tregs in the tumor environment is indicative of a poor prognosis. Tregs are thought to suppress tumor immunity, hindering the body's innate ability to control the growth of cancerous cells. There are different sub-populations of regulatory T cells, including those that express CD4, CD25, and Foxp3 (CD4+CD25+ regulatory T cells). These "naturally- occurring" Tregs are different from helper T cells and are also distinguishable from "suppressor" T cell populations that are generated in vitro. [0811] While regulatory T cells are crucial in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance, they are thought to contribute to the progress of many tumors. Most tumors elicit an immune response in the host that is mediated by tumor antigens, thus distinguishing the tumor from other non-cancerous cells. As cancer cells express both self- and tumor-associated antigens, Tregs are key to dampening effector Tcell responses, and therefore represent one of the main obstacles to effective anti-tumor response and the failure of current therapies that rely on induction or potentiation of anti-tumor responses. Thus, controlling the function of these Tregs cells in the tumor microenvironment without compromising peripheral tolerance represents a useful cancer therapy.

[0812] Tregs seem to be preferentially trafficked to the tumor

microenvironment. While Tregs normally make only about 4% of CD4+ T Cells, they can make up as much as 20-30% of the total CD4+ population around the tumor microenvironment. It is widely recognized that the ratio of Tregs to Teffectors in the tumor microenvironment is a determining factor in the success the immune response against the cancer. High levels of Tregs in the tumor microenvironment are associated with poor prognosis in many cancers, such as ovarian, breast, renal, and pancreatic cancer, indicating that Tregs suppress Teffector cells and hinder the body's immune response against the cancer.

[0813] The tryptophan (TRP) to kynurenine (KYN) metabolic pathway is established as a key regulator of innate and adaptive immunity. Several preclinical models suggest that this immune tolerance pathway is active in cancer immunity, autoimmunity, infection, transplant rejection, and allergy. Drugs targeting this pathway, e.g, indoleamine-2,3-dioxygenase (IDO), are in clinical trials with the aim at reversing cancer-induced immunosuppression. In certain embodiments, the disclosure provides genetically engineered bacteria which can metabolize and/or produceTRP and/or KYN using the cassettes described herein. Induction of these metabolic functions may be regulated by environmental conditions and any of the regulatory mechanisms described herein, e.g. regulation of the gene cassette expression through inducible promoters described herein.

[0814] The catabolism of tryptophan is a central pathway maintaining the immunosuppressive microenvironment in many types of cancers. Tumor cells or myeloid cells in the tumor microenvironment express high levels of indoleamine-2,3- dioxygenase 1 (IDOl), which is the first and rate- limiting enzyme in the degradation of tryptophan. This enzymatic activity results in the depletion of tryptophan in the local microenvironment and subsequent inhibition of T cell responses, which results in immunosuppression (as T cells are particularly sensitive to low tryptophan levels). More recent preclinical studies suggest an alternative route of tryptophan degradation in tumors via the enzyme TRP-2,3-dioxygenase 2 (TDO). Thus, tumor cells may express and catabolize tryptophan via TDO instead of or in addition to IDOl .

[0815] In addition, several studies have proposed that immunosuppression by tryptophan degradation is not solely a consequence of lowering local tryptophan levels but also of accumulating high levels of tryptophan metabolites. Preclinical studies and analyses of human tumor tissue have demonstrated that T cell responses are inhibited by tryptophan metabolites, primarily by binding to the aryl hydrocarbon receptor (AHR), a cytoplasmic transcription factor. These studies show that binding of the tryptophan metabolite kynurenine and other metabolites to the aryl hydrocarbon receptor results in reprogramming the differentiation of naive CD4+ T-helper (Th) cells favoring a regulatory T cells phenotype (Treg) while suppressing the differentiation into interleukin- 17 (IL- 17)-producing Th (Thl7) cells. Activation of the aryl hydrogen receptor also results in promoting a tolerogenic phenotype on dendritic cells.

[0816] In some embodiments, the genetically engineered bacteria are useful in the treatment, prevention and/or management of cancer, including but not limited to one or more of the cancers described herein. In some embodiments, the genetically engineered bacteria may counteract immunosuppression though expression of one or more of the circuits described herein. In some embodiments, the genetically engineered bacteria may shift T cell phenotypes from the regulatory phenotype to the Thl7 phenotype. In some embodiments, the genetically engineered microorganisms of the present disclosure, e.g., genetically engineered bacteria or genetically engineered oncolytic viruses are capable of depleting Tregs or inhibiting or blocking the activation of Tregs by producing tryptophan. In some embodiments, the genetically engineered microorganisms of the present disclosure capable of increasing the CD8+: Treg ratio (e.g., favors the production of CD8+ over Tregs) by producing tryptophan. As discussed above, studies have shown that the binding of kynurenine to the aryl hydrocarbon receptor results in the production of regulatory T cells (Tregs). Thus, in some embodiments, the genetically engineered bacteria or genetically engineered oncolytic viruses comprise a mechanism for metabolizing or degrading kyurenine. In some embodiments, the genetically engineered bacteria or genetically engineered oncolytic viruses comprise sequence encoding the enzyme kynureninase. In some embodiments, the genetically engineered bacteria or genetically engineered oncolytic viruses comprise gene sequence(s) encoding enzymes of the tryptophan biosynthetic pathway and sequence encoding kynureninase. The disclosure also provides strains of interest with increased kynurenine metabolism and uptake and lowered TRP uptake, which have enhanced abililty for reducing immune suppression. As a result, this strain has improved therapeutic properties in a number of applications, including but not limited to immunoncology.

[0817] In some embodiments, the genetically engineered bacteria or genetically engineered oncolytic viruses comprise sequence encoding the enzyme kynureninase and a secretion mechanism and corresponding gene sequence(s) encoding the secretion system, such that kynureninase is secreted into the extracellular environment.

[0818] In some embodiments, the genetically engineered bacteria can be combined with one or more conventional or other treatment described herein or known in the art for the treatment, prevention and/or management of cancer, including but not limited to checkpoint inhibitors and/or chemotherapy.

Autoimmune Disease and Gut Barrier Function

[0819] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of autoimmune disease, including, but not limited to the autoimmune diseases described herein.

[0820] The expression of IDO and kynurenine production by different cell types, as well as the capacity of IDO competent regulatory dentritic cells to induce Treg cells through high levels of kynurenine production, could have broader immunological significance in tolerance and immunoregulation, e.g. , it has been suggested that defects in the immunoregulatory mechanism initiated by IDO and the kynurenine pathway are involved in the development of autoimmune conditions, such as multiple sclerosis and autoimmune diabetes.

[0821] In addition to genetic susceptibility, making the individual react abnormally to self antigens, compromised gut barrier function plays a central role in autoimmune disease pathogenesis (Lerner et al., 2015a; Lerner et al., 2015b; Fasano et al., 2005; Fasano, 2012). A single layer of epithelial cells separates the gut lumen from the immune cells in the body. The epithelium is regulated by intercellular tight junctions and controls the equilibrium between tolerance and immunity to nonself- antigens (Fasano et al., 2005). Disrupting the epithelial layer can lead to pathological exposure of the highly immunoreactive subepithelium to the vast number of foreign antigens in the lumen (Lerner et al., 2015a) resulting in increased susceptibility to both intestinal and extraintestinal autoimmune disorders (Fasano et al., 2005). Some foreign antigens are postulated to resemble self-antigens and can induce epitope- specific cross- reactivity that accelerates the progression of a pre-existing autoimmune disease or initiates an autoimmune disease (Fasano, 2012). Not only gastrointestinal autoimmune disorders like IBD and celiac disease, but also systemic autoimmune diseases like rheumatoid arthritis and type I diabetes, are autoimmune disorders that are thought to involve increased intestinal permeability or "leaky gut" (Lerner et al., 2015b), and defects in components of the epithelial barrier are etiologic factors in the pathogenesis of inflammatory bowel diseases (IBDs) (Pastorelli et al., Front Immunol. 2013; 4: 280; Central Role of the Gut Epithelial Barrier in the Pathogenesis of Chronic Intestinal Inflammation: Lessons Learned from Animal Models and Human Genetics).

[0822] As used herein "leaky gut" or "leaky gut syndrome syndrome" refers to an increased intestinal permeability, e.g.,, which can occur in patients susceptible to a number of dieases including IBD, celiac disease, type 1 diabetes and other metabolic and/or autoimmune diseases described herein. For example, bacterial dysbiosis, long- term antibiotics use, or susceptibility to intestinal inflammatory disease is associated with "leaky gut" as a pathogenic cause, which also further promotes disease onset and progression.

[0823] In individuals who are genetically susceptible to autoimmune disorders, dysregulation of intercellular tight junctions can lead to disease onset (Fasano, 2012). In fact, the loss of protective function of mucosal barriers that interact with the environment is necessary for autoimmunity to develop (Lerner et al., 2015a).

[0824] Changes in gut microbes can alter the host immune response (Paun et al., 2015; Sanz et al., 2014; Sanz et al., 2015; Wen et al., 2008). For example, in children with high genetic risk for type 1 diabetes, there are significant differences in the gut microbiome between children who develop autoimmunity for the disease and those who remain healthy (Richardson et al., 2015). Thus, enhancing barrier function and reducing inflammation in the gastrointestinal tract are potential therapeutic mechanisms for the treatment or prevention of autoimmune disorders.

[0825] For example, leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate. In addition, a number of trptophan metabolites, including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehhyde, indole-3 propionic acid, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown to be essential for gut homeostais and promote gut-barrier health. These metabolites bind to aryl hydrocarbon receptor (Ahr). After agonist binding, AhR translocates to the nucleus, where it forms a heterodimer with AhR nuclear translocator (ARNT). AhR-dependent gene expression includes genes involved in the production of mediators important for gut homeostasis; these mediators include IL-22,

antimicrobicidal factors, increased Thl7 cell activity, and the maintenance of intraepithelial lymphocytes and RORyt-i- innate lymphoid cells.

[0826] Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3-dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis.

[0827] In addition, some indole metabolites, e.g., indole 3-propionic acid (IP A), may exert their effect an acitvating ligand of Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function, through downregulation of TLR4 signaling (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). As a result, indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health.

[0828] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune disorders described herein. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein.

[0829] Thus, in some embodiments, the genetically engineered bacteria of the disclosure produce tryptophan and/or one or more or more tryptophan metabolites for the treatment of autoimmune disorders. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of tryptophan and/or one or more tryptophan metabolites for the improvement of gut barrier health. In some embodiments, , the genetically engineered bacteria provide metabolites, which improve AhR agonism. In some embodiments, the genetically engineered bacteria provide metabolites which improve PXR agonism.

[0830] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of autoimmune diseases comprise one or more gene(s) or gene sequence(s) for the production of kynurenine, kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

Table 30. Diseases related to intestinal permeability

functional GI diseases

Inflammatory bowel disease, Chronic inflammation (e.g. arthritis)

Celiac disease

Cancer (esophagus, Obesity-associated metabolic diseases

colorectal) (NASH, diabetes type I and II, CVD)

Chronic Viral and Bacterial Infections

Trp catabolites, by activating aryl hydrocarbon receptor (AhR), play an important role in antimicrobial defense and immune regulation. IDO/AhR acts as a double-edged sword by both depleting L-Trp to starve the invaders and by contributing to the state of immunosuppression with microorganisms that were not cleared during acute infection.

Additionally, by inducing serotonin depletion, the KP has become recognized as a key player in the pathogenesis of several major neuroinflammatory brain conditions associated with chronic viral infections such as HIV, cytomegalovirus (CMV), and HSV (Kandanearatchi A, Brew BJ. The kynurenine pathway and quinolinic acid: pivotal roles in HIV associated neurocognitive disorders. FEBS J. 2012;279(8): 1366-74; adeghi M, Lahdou I, Daniel V, et al. Strong association of phenylalanine and tryptophan metabolites with activated cytomegalovirus infection in kidney transplant recipients. Hum Immunol. 2012;73(2): 186-92.).

In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of chronic viral and bacterial infections described herein. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the chronic viral and bacterial infections herein.

[004] In some embodiments, the genetically engineered bacteria of the present invention are useful for the treatment of one or more viral and/or bacterial infections, e.g. through the local or systemic modulation of tryptophan metabolite ratios. In some embodiments, the genetically engineered bacteria modulate serotonin levels, e.g., increase or decrease blood serotonin levels. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) for the production of serotonin from tryptophan. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of serotonin.

[005] In some embodiments, the genetically engineered bacterial

compositions can be combined with any conventional or other treatment(s) described herein or known in the art for the treatment, prevention and/or management of viral and/bacterial infections. It is understood that any such additional treatment would not be toxic to the genetically engineered bacteria.

Human Immunodeficiency Virus

[006] In some embodiments, the genetically engineered bacteria are useful in the treatment, prevention and/or management of human immunodeficiency virus (HIV) infec tion. HIV is a lentivirus spread through body fluids and which attacks the immune system though the infection of CD4+ T cells, macrophages, and dendritic cells. If left untreated, HIV can lead to acquired immunodeficiency syndrome (AIDS), a failure of the immune system to battle opportunistic infections or cancer.

[007] CD4 T-cell depletion and chronic immune activation are hallmarks of

HIV infection. Persistent immune activation despite suppressive antiretroviral therapy (ART) is associated with an increased risk of AIDS and non-AIDS related events, including cardiovascular, liver and kidney diseases, cancers, and alteration of neurocognition. The persistent immune activation is caused by the ability of HIV to alter the gastrointestinal environment, leading to changes in gut microbiota and mucosal permeability, which results in dysregulation of the intestinal immune barrier, translocation of immuno stimulatory microbial products contributing to systemic immune activation.

[008] IDO is highly expressed in gut mucosa during initial HIV infection, leading to increased levels of kynurenine, which may function to alter the local ratio of Tregs/TH17 cells by promoting Tregs and attenuating protective TH17 responses (Favre D, et al. Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease. Sci Transl Med. 2010;2:32ra36).

[009] In addition to the kynurenine produced by the gut mucosa, gut-resident bacteria with capacity to metabolize tryptophan through the kynurenine pathway were found to be enriched in HIV-infected subjects. The accumulation of these bacteria strongly correlated with kynurenine levels in HIV-infected subjects, and the study showed that the bacteria were capable of kynurenine production in vitro (Vujkovic-Cvijin et al. Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism. Sci Transl Med. 2013 Jul 10; 5(193): 193ra91), suggesting that the outgrowth of this community may be driven by the persistent host IDOl activity found during chronic HIV infection.

[010] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of HIV infection. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of HIV infection.

[011] In some embodiments, the genetically engineered bacteria are employed to target and replace the HIV-specific dysbiotic gut bacteria to reverse HIV- associated immunopathology. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) described herein to reduce kynurenine levels. In some embodiments, the genetically engineered bacteria are useful for the treatment, prevention and/or management of HIV infection. In some embodiments, the genetically engineered bacteria of the present disclosure counteract the immune suppressive environment upon HIV infection. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s), which when expressed, cause a shift from Tregs to Thl7 cells. In some embodiments, the bacteria comprise one or more gene sequence(s) encoding one more tryptophan biosynthesis enzymes, alone or in combination with one or more gene sequence(s) encoding one more enzymes described herein for the degradation of kynurenine. In some embodiments, the genetically engineered bacteria comprise transporters described herein, which facilitate the uptake of kynurenine and/or tryptophan from the environment. The disclosure also provides strains of interest with increased kynurenine and lowered TRP uptake, which have enhanced ability for reducing immune suppression.

[0831] Additionally, two Trp metabolites, 3-HK and QUIN(Quin), can be detected in the cerebrospinal fluid (CSF) of HIV-infected patients and are correlated with the severity of HIV-associated neurocognitive disorder (HAND) and infection of myeloid-derived cells in the brain (The kynurenine pathway and quinolinic acid: pivotal roles in HIV associated neurocognitive disorders. (Kandanearatchi A, Brew BJ FEBS J. 2012 Apr; 279(8): 1366-74.). In some embodiments, the genetically engineered bacteria described herein comprise a gene cassette described herein which reduces levels of kynurenine available for 3-HK and QUIN(Quin) production. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) encoding enzymes which can metabolize 3-HK and/or QUIN.

[0832] Finally, gut epithelial barrier dysfunction, innate immune activation, inflammation, and coagulation of T cells, independently predict mortality in individuals with treated HIV infection with a history of AIDS and are viable targets for

interventions (see, e.g., Hunt et al., J Infect Dis. 2014 Oct 15;210(8):1228-38; Gut epithelial barrier dysfunction and innate immune activation predict mortality in treated HIV infection). As such, in some embodiments, the genetically engineered bacteria for the treatment of HIV, may comprise one or more gene sequences which produce anti- inflammatory tryptophan metabolites, e.g., indole metabolites and/or kynurenine. In some

embodiments, the genetically engineered bacteria also comprise gene sequence(s) encoding enzyme(s) for the production of tryptophan, or transporters for the import of tryptophan. Such circuits may also be combined with gut barrier enhancer circuits, such as SCFA, e.g., butyrate, producing circuits referenced elsewhere herein.

Viral Hepatitis

[012] In some embodiments, the genetically engineered bacteria are useful in the treatment, prevention and/or management of viral hepatitis. IDO induction in chronic viral infections is considered to be the main cause of the decreased serum Trp levels. Cozzi et al studied patients chronically infected by HCV or hepatitis B virus (HBV) who were found to have lower serum Trp concentrations than healthy volunteers. Furthermore, Comai et al confirmed the decrease of Trp in HCV-infected patients as well as a decline of serotonin pathway, contributing to the development of depressive symptoms in HCV patients undergoing IFN-a therapy. (Comai S, Cavalletto L, Chemello L, et al. Effects of PEG-interferon alpha plus ribavirin on tryptophan metabolism in patients with chronic hepatitis C. Pharmacol Res. 2011;63(l):85-92). Using primary human hepatocytes, Lepiller et al showed that HCV infection stimulates IDO expression and concurred with the expression of types I and III IFNs and IFN- stimulated genes (Lepiller Q, Soulier E, Li Q, et al. Antiviral and immunoregulatory effects of indoleamine-2,3-dioxygenase in hepatitis C virus infection. J Innate Immun. 2015). These study findings showed that HCV infection directly induced IDO and IFN expression (Comai S, Cavalletto L, Chemello L, et al. Effects of PEG-interferon alpha plus ribavirin on tryptophan metabolism in patients with chronic hepatitis C. Pharmacol Res. 2011;63(l):85-92).

[013] High plasma KYN:TRP ratios have been reported in association with increased IDO expression in hepatocytes and DCs infected with HBV and HCV.

Higashitani et al demonstrated that KYN levels correlated with advanced liver conditions such as fibrosis. Systemic effects resulting from induction of KP have been reported in monocytes isolated from PBMCs obtained from HCV-positive patients. When activated with LPS or INF-γ, these cells were shown to differentiate into IDO- expressing DCs capable of a more potent Treg induction (Association of enhanced activity of indoleamine 2,3-dioxygenase in dendritic cells with the induction of regulatory T cells in chronic hepatitis C infection. Higashitani K, et al. , J Gastroenterol. 2013 May; 48(5):660-70.).

[014] In some embodiments, the genetically engineered bacteria of the present disclosure counteract the immune suppressive environment upon HBV and/or HCV infection in the treatment, prevention and/or management of HBV and/or HCV infection. In some of the embodiments, a shift from Tregs to Thl7 cells may occur in response to the genetically engineered bacteria, as described elsewhere herein.

[015] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of viral hepatitis. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of viral hepatitis.

[016] In some embodiments, the genetically engineered bacteria comprise any of the cassettes described herein to reduce kynurenine levels and/or increase tryptophan levels. In some embodiments, strains are created using ALE with increased

kynurenenine uptake and/or consumption activity and lowered TRP uptake, which have enhanced abililty for reducing immune suppression. As a result, this strain has improved therapeutic properties in a number of applications, including but not limited to, the treatment, prevention and/or management of HB V and/or HCV infection.

[017] Intestinal barrier damage is associated with HCV-related liver cirrhosis.

Disrupted gut barrier leads to an increased passage of microbial products and to an activation of the mucosal immune system and secretion of inflammatory mediators, which in turn might increase barrier dysfunction. As such, in some embodiments, the genetically engineered bacteria for the treatment of viral hepatitis, e.g., HCV, may comprise one or more gene sequences which produce ant i- inflammatory tryptophan metabolites, e.g., indole metabolites and/or kynurenine. In some embodiments, the genetically engineered bacteria also comprise gene sequence(s) encoding enzyme(s) for the production of tryptophan, or transporters for the import of tryptophan. Such circuits may also be combined with gut barrier enhancer circuits, such as SCFA, e.g., butyrate, producing circuits referenced elsewhere herein.

CMV and EBV

[018] In some embodiments, the genetically engineered bacteria are useful in the treatment, prevention and/or management of CMV and/or EBV.

[019] Human infection with CMV, another member of the Herpesviridae family, also persists for life by counteracting IFN-mediated antiviral defense.

[020] Infectious mononucleosis is the most common clinical manifestation of infection with Epstein-Barr virus (EBV), another widely spread herpesvirus family member that is also associated with malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma. EBV-induced IDO expression of macrophages suppressed T-cell proliferation, impaired the cytotoxic activity of CD8 T-cells. IDO induction during chronic active EBV infection is also associated with decreased serotonin levels leading to symptoms, including mood disturbances. All these observations point to the contribution of the KP on disease tolerance.

[021] In some embodiments, the genetically engineered bacteria of the present disclosure counteract the immune suppressive environment upon CMV or EBV infection for in the treatment, prevention and/or management of CMV or EBV infection. In some of the embodiments, a shift from Tregs to Thl7 cells may occur in response to the genetically engineered bacteria, as described elsewhere herein.

[022] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of CMV and EBV infection. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of CMV and EBV infection.

[023] In some embodiments, the genetically engineered bacteria comprise any of the cassettes described herein to reduce kynurenine levels and/or increase tryptophan levels. In some embodiments, strains are created using ALE with increased

kynurenenine uptake and/or consumption activity and lowered TRP uptake, which have enhanced abililty for reducing immune suppression. As a result, this strain has improved therapeutic properties in a number of applications, including but not limited to, the treatment, prevention and/or management of CMV or EBV infection.

Herpes virus

[024] Human herpes simplex virus type 1 (HSV-1) and HSV-2 are members of the Herpesviridae family, which establish latency in neural ganglia. HSV-2 is the primary cause of genital herpes lesions and establishes a lifelong latent infection in the neurons of the sacral ganglia, which can be reactivated depending on the host immune response. IFN-γ production remains a key element of defense against HSV infection, capable of inhibiting virus replication. Here, IFN-y-induced IDO activity acts as a potent antiviral effector mechanism against HSV-2 infection and excess TRP is capable of abrogating the antiviral effect of IFN-γ (Adams O, Besken K, Oberdorfer C,

MacKenzie CR, Russing D, Daubener W. Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase. Microbes Infect. 2004;6(9):806-12.) Infection of mice by herpes simplex virus type 1 raised the levels of quinoimic acid in mice, in parallel with paralysis, in some embodiments, the genetically engineered bacteria are useful in the treatment, prevention and/or management of herpes virus.

[025] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment herpes virus infection. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of herpes virus infection.

[026] In certain embodiments, the genetically engineered bacteria comprise a circuit described herein to degrade KYN to AA in the treatment, prevention and/or management of herpes virus infection. Wishout wishing to be bound by theory, this shifts the KP away from other downstream metabolites. In some embodiments, the genetically engineered bacteria can be combined with one or more additional conventional or other treatment(s) described herein or known in the art for the treatment, prevention and/or management of herpes virus.

Parasitic infection

[027] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of parasitic infection. Infections with parasites also lead to IDO-mediated production of kynurenines. A study of patients with cerebral malaria caused by an infection with Plasmodium falciparum revealed an increase in QUIN, KYNA and PA levels in CSF. Levels of QUIN and PA were associated with a higher rate of convulsions and a fatal outcome.

[028] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of parasitic infection. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of parasitic infection.

[029] In some embodiments, the genetically engineered bacteria comprise genetic circuits described herein, including but not limited to circuits, which allow the conversion of KYN to AA. Without wishing to be bound by theory, through the production of AA, available is siphoned away from other KP branches involved in the production of QUIN, KYN A, and PA.

Autoimmune, Metabolic, and Cardiovascular Disorders

Rheumatoid Arthritis and Systemic Lupus Erythematosus

In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Rheumatoid Arthritis (RA). RA is an inflammatory autoimmune systemic disease affecting around 1 % of the western population and that principally targets synovial joints. The disease is usually progressive and results in swelling of joints, pain and stiffness, with ankylosis developing in many cases. The disease involves inflammation of the capsule surrounding the joints, hyperplasia of synovial cells, oedema and fibrosis. The pathology of the disease process frequently causes destruction of articular cartilage and ankylosis of the joints. Immune system activation and the production of cytokines are known to play a crucial rolel in RA. In RA, activity of proinflammatory Thl7 cells overtake the ant i- inflammatory activity of Treg cells as chronic inflammation of the joints progress.

RA is associated with increased production of kynurenine, which is thought to have an ant i- inflammatory effect. In one study in RA patients, the kynurenine/tryptophan ratio was higher in RA patients than in controls, and the levels of kynurenine, as well as the kynurenine/tryptophan ratio, correlated with levels of neopterin (a marker of immune cell activation), indicating that the kynurenine pathway was activated in these patients (Schroecksnadel K, Kaser S, Ledochowski M, et al. Increased degradation of tryptophan in blood of patients with rheumatoid arthritis. J Rheumatol. 2003

Sep;30(9): 1935-9). An ant i- inflammatory role for kynurenine in RA has been illustrated in several studies in a collagen induced arthritis (CIA) mouse model of RA. In IDOl- KO mice with CIA, the incidence of the disease was found to be higher and the severity of the symptoms were greater than in the wild type (WT) CIA mice (Kolodziej Investigation of the kynurenine pathway in Indoleamine 2, 3 dioxygenase deficient mice with inflammatory arthritis Transgenic Res. 2013; 22(5): 1049-1054 and references therein). In another study, the progression of CIA in mice treated with L-kynurenine significantly reduced both clinical and histological progression of arthritis as compared to controls (Criado G, Simelyte E, Inglis JJ, Essex D, Williams RO. Indoleamine 2,3 dioxygenase-mediated tryptophan catabolism regulates accumulation of Thl/Thl7 cells in the joint in collagen- induced arthritis. Arthritis Rheum. 2009 May;60(5): 1342-1351.

[030] Altered gut microbiota have been shown to play a pathogenic role in the development of RA (See e.g., Wu HJ, Ivanov, Darce J, Hattori K, Shima T, Umesaki Y, et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity. 2010;32:815-827). The involvement of intestinal changes in the pathogenesis of RA was suggested by findings of increased intestinal permeability and the presence of gastrointestinal symptoms in patients with juvenile idiopathic arthritis. Moreover, the frequent occurrence of arthritis in patients suffering from IBD suggests participation of the gut in this immune mediated rheumatic disorder (Tlaskalova-Hogenova et al., Cell Mol Immunol. 2011 Mar; 8(2): 110-120; The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ- free and gnotobiotic animal models of human diseases, and references therein).

[031] SLE is characterized by autoantibodies against self-antigens, resulting in inflammation-mediated multiorgan damage. One study describes the observation of a decreased percentage of IDO-expressing peripheral cells iRA and iSLE patients as compared to controls, which may play a critical role in tolerance loss in these diseases (Furuzawa-Carballeda et al., Indoleamine 2,3-dioxygenase-expressing peripheral cells in rheumatoid arthritis and systemic lupus erythematosus: a cross-sectional study;

European Journal of Clinical Investigation Vol 41).

[0833] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., RA and/or SLE. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., RA and/or SLE.

[0834] In certain embodiments, the genetically engineered bacteria described herein comprise circuits which reduce inflammation, e.g. , through the removal of tryptophan from the circulation and the generation of KYN and/or KYNA and/or indole metabolites described herein.

[0835] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of RA comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

[0836] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of SLE comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein. Psoriasis

[0837] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Rheumatoid Psoriasis. Psoriasis is a common skin disorder that forms thick, red, bumpy patches covered with silvery scales. At the root of the disease is a dysregulation of the immune system, which causes inflammation, triggering new skin cells to form too quickly.

Normally, skin cells are replaced every 28 to 30 days, however with psoriasis, new cells grow every three to four days. The buildup of old cells being replaced by new ones creates silver scales.

[0838] Higher levels of kynurenine are found in psoriasis patients compared to healthy subjects. However, a lower IDO activity in those patients with higher Psoriasis Area and Severity Index was observed, indicating an ant i- inflammatory role for kynurenine and/or the kynurenine pathway. Kynureninase expression positively correlates with disease severity and inflammation and is reduced on successful treatment of psoriasis or AD. (Harden et al, J Allergy Clin Immunol. 2015 Dec 22. pii: S0091- 6749(15)01585-7. The tryptophan metabolism enzyme L-kynureninase is a novel inflammatory factor in psoriasis and other inflammatory diseases).

[0839] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., psoriasis. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., psoriasis.

[0840] In some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites for the treatment, management and/or prevention of psoriasis. In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of psoriasis comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the

extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

Graft vs Host Disease

[0841] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Graft vs host disease. Wider application of allogeneic bone marrow transplantation (BMT) is restricted due to graft-versus-host disease (GVHD), the most significant cause of morbidity and mortality after transplantation. Intestinal barrier loss has a critical role in the pathogenesis of graft-versus-host disease (GVHD), and its role in initiating and establishing a pathogenic inflammatory cycle in GVHD is becoming evident. The luminal microbiome contributes to the pathogenesis of GVHD, as germ-free mice are at least partially protected and antibiotics can provide benefit in human subjects (see, e.g., Nalle et al., Sci Transl Med. 2014 Jul 2; 6(243): 243ra87; Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host

disease). Iintestinal damage, including barrier loss induced by pre-transplant conditioning allows translocation of gut microbiota and microbial products that stimulate GVHD development, and progression (Nalle et al., 2014).

[0842] Uncontrolled spreading of commensal bacteria and tissue injury further stimulates donor alloreactive T cells, and thereby aggravates tissue damage in the gut, skin, and liver, leading to acute GVHD within three months of transplantation. Major characteristics of acute GVHD are activated antigen-presenting cells (APC), proinflammatory cytokine milieu, and enhanced recruitment and activation of effector T cells. Gastrointestinal acute GVHD or mucositis is a key contributor to post-transplant morbidity and mortality, since it cannot be efficiently managed through

immunosuppressive therapy. The colonic mucosal tissue alterations of GVHD patients are comparable to IBD (Nalle and Turner, Intestinal barrier loss as a critical pathogenic link between inflammatory bowel disease and graft-versus-host disease;

Mucosallmmunology I VOLUME 8 NUMBER 4 1720-730).

[0843] Additionally, an important function for kynureine during GVHD has been described. - Without host IDO expression, mice experience markedly more severe disease (Jasperson LK, Bucher C, Panoskaltsis-Mortari A, et al. Indoleamine 2,3- dioxygenase is a critical regulator of acute graft-versus-host disease lethality. Blood. 2008; 111:3257-65). The results from this study indicate that enhanced IDO activity or induction of IDO before transplantation may suppress disease and the authors speculate that administration of tryptophan catabolites may mimic IDO activity and also be able to dampen disease. Thus, in some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites, e.g., for the prevention, management, or treatment of GVHD.

[0844] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., GVHD. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., GVHD.

[0845] In some embodiments, the genetically engineered bacteria produce one or more gut barrier enhancer molecules and/or anit- inflammatory molecules for the prevention, treatment and/or management of GVHD. For example, a number of trptophan metabolites, in additiona to kynurenine and kynurenic acid, termed indoles, such as indole-3 aldehhyde, indole-3 propionic acid, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown to be essential for gut homeostais and promote gut-barrier health.

[032] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of GVHD comprise one or more gene(s) or gene sequence(s) which modulate systemic tryptophan and/or tryptophan metabolite availability. IBP and colitis, and Crohns disease

[0846] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of inflammatory bowel diseases (IBDs) and/or colitis and/or Crohn's disease. Idiopathic IBD, Crohn's disease and ulcerative colitis are severe chronic disorders affecting approximately 0.2% of the human population. Inflammatory IBDs are a group of diseases characterized by significant local inflammation in the gastrointestinal tract driven by T cells and activated macrophages and by compromised function of the epithelial barrier that separates the luminal contents of the gut from the host circulatory system (Ghishan et al., 2014). IBD pathogenesis is linked to both genetic and environmental factors and may be caused by altered interactions between gut microbes and the intestinal immune system.

[0847] In the gut, IDOl expressed in the lamina propria is one of the most highly upregulated genes in human IBD and animal models of colitis. Disease severity was worsened in mice receiving the IDO inhibitor 1-DL-methyl tryptophan (lmT), suggesting that certain KP metabolites down-regulate Thl inflammatory responses within the intestinal tract (Gurtner GJ, Newberry RD, Schloemann SR, et al. Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic acid colitis in mice. Gastroenterology. 2003; 125: 1762-73.), likely through kynurenine and/or kynurenic acid mediated Ahr activation. Increased IDO activity corresponds to the increases in kynurenine levels observed in clinical studies. For example, a study comparing serum concentrations of kynurenines in patients with mild inflammatory bowel disease (subdivided into subgroups of those with Crohn's disease and those with ulcerative colitis) and in sex- and age-matched control subjects found normal tryptophan concentrations in all patients but significantly elevated levels of the kynurenine in IBD patients regardless of the subgroup. (Forrest et al., Purine, Kynurenine, Neopterin and Lipid Peroxidation Levels in Inflammatory Bowel Disease; J Biomed Sci 2002;9:436- 442).

[0848] In addition to kynurenine pathways metabolites, other metabolites generated through pathways involved in the metabolism of tryptophan and indole influence epithelial barrier integrity and the presence of an inflammatory or tolerogenic environment in the intestine and beyond as described elsewhere herein, e.g., through Ahr and PXR agonism. For example, indole- 3 -aldehyde from dietary tryptophan by the microbiota induces the production of IL-22, a protective cytokine during colon inflammation, from innate lymphoid cells (ILCs) in colitis models (see, e.g., McCarville et al., Novel perspectives on therapeutic modulation of the gut microbiota Therap Adv Gastroenterol. 2016 Jul; 9(4): 580-593). In another example, studies in a mouse model of colitis showed that indol-3-propionic acid (IP A), promotes immune homeostasis and gut barrier function in the intestinal mucosa during inflammatory conditions and prevents IBD. In this study, expression of IL-22 by intraepithelial lymphocytes (IELs) of the small intestine increased, and a PXR dependent increase in IL-17A and IFN gamma expressing IELS was observed (Romagnoli et al., The Journal of Immunology, 2016, vol.196 (1 Supplement) 67.10; Commensal metabolite indol-3-propionic acid promotes gut barrier function by regulating IL-22 production during intestinal inflammatory conditions).

[0849] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., IBD, colitis, or Crohns disease. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., IBD, colits, or Crohns disease.

[0850] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of IBDs, including but not limited to IBD, colitis, and Crohn's disease, comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the outside the bacterial cell is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

Celiac disease

[0851] Celiac disease is a genetic chronic immune-mediated enteropathy that is triggered by dietary wheat gluten or related prolamins, characterized by increased cellularity (intraepithelial lymphocytes) and atrophy of jejunal mucosa. The

autoimmune nature of this disease was confirmed by the presence of autoimmune mechanisms directed against several autoantigens, including the most diagnostically important autoantigen, tissue transglutaminase. The frequent association between celiac disease and other autoimmune diseases, particularly type 1 diabetes (T1D) and thyroiditis, suggests that celiac enteropathy shares certain pathogenic mechanisms with other autoimmune diseases. Viral infections, including adenovirus, hepatitis C virus, and rotavirus and bacterial infections, along with nutritional factors and toxins, have been found to cause or enhance mucosal responses to gluten in the gut, and may play a role in the pathogenesis of this celiac disease.

[0852] Gut mucosal barrier dysfunction has been demonstrated and confirmed by genetic studies in patients with celiac disease (Tlaskalova-Hogenova et al 2011, Cell Mol Immunol. 2011 Mar; 8(2): 110-120. and references therein). As such, enhancing gut barrier function, e.g., through providing the engineered bacteria provided herein, is a useful approach for the treatment of celiac disease.

[0853] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., celiac disease. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., celiac disease.

[0854] In some embodiments, the genetically engineered bacteria produce a gut barrier enhancer or an anti- inflammatory effector, e.g., an Ahr agonist. In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of celiac disease, comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the

extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

Colorectal cancer and Cachexia

[0855] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of colorectal cancer (CRC). CRC is the third most common cancer worldwide, and in the United States is the secondleading cause of cancer related death (almost 50,000 per year). In most cases, the transition from normal colon epithelium to cancer is influenced by the acquisition of somatic mutations and environmental factors including diet and lifestyle. The adenomatous polyposis coli (APC) gene is a key component to Wnt signaling and is mutated in most CRCs. Chronic inflammation is also a risk factor for CRC. Colitis- associated cancer (CAC) is a form of CRC that develops in patients with chronic inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. Although most CRC develops in patients without underlying inflammatory bowel disease, 'inflammatory signature' genes characteristic of colitis-associated cancer are also upregulated in these colorectal cancers. The mechanism by which chronic intestinal inflammation leads to CRC most likely involves inflammatory cells and their associated mediators such as interleukin-6 (IL-6), tumor necrosis factor-a (TNF- a), IL-23, and reactive oxygen species form a microenvironment favoring the development of CRC.

[0856] Altered gut microbiota are a hallmark of CRC in human patients. In mice, colitis can promote tumorigenesis by altering microbial composition and inducing the expansion of microorganisms with genotoxic capabilities. At the same time, inflammation promotes barrier dysfunction by suppression of protective

mucin/antimicrobial peptide production, and enhances the contact of the bacteria with the colonic mucosa (see e.g., Jenkins, Cancer Cell (2016) 29 615-617, and references therein). Addtionally, Grivennikov et al. (2013) showed that barrier deterioration induced by colorectal-cancer- initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth (Nature. 2012 November 8; 491(7423): 254-258; Adenoma- linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

[0857] Cachexia is a condition characterized by severe wasting of muscle and adipose tissue and is a common complication in late-stage cancers, in particular gastrointestinal cancers.

[0858] Efforts to preserve the integrity of the gut epithelial barrier and/or limit intestinal inflammation in cancer patients may help avoid the serious metabolic alterations associated with cachexia. Multimodal treatment strategies that include interventions aimed at maintaining gut barrier function and correcting dysbiosis may be used to in controlling cachexia. Therefore, strategies reducing intestinal inflammation and enhancing gut barrier function are useful therapeutic approaches for CRC.

[0859] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of colorectal cancer and/or cachexia. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of colorectal cancer and/or cachexia.

[0860] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of CRC and/or cachexia, comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

Insulin Resistance, and Diabetes Type I and Type II

[0861] Diabetes mellitus type 1 (also known as type 1 diabetes) is a form of diabetes mellitus that results from the autoimmune destruction of the insulin-producing beta cells in the pancreas. The subsequent lack of insulin leads to increased glucose in blood and urine. The classical symptoms are frequent urination, increased thirst, increased hunger, and weight loss. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of diabetes mellitus.

[0862] Diabetes mellitus type 2 is a long term metabolic disorder that is characterized by high blood sugar, insulin resistance, and relative lack of insulin.

Common symptoms include increased thirst, frequent urination, and unexplained weight loss. Symptoms may also include increased hunger, feeling tired, and sores that do not heal. Often symptoms come on slowly. Long-term complications from high blood sugar include heart disease, strokes, diabetic retinopathy which can result in blindness, kidney failure, and poor blood flow in the limbs which may lead to amputations.

[0863] Insulin resistance (IR) is generally regarded as a pathological condition in which cells fail to respond to the normal actions of the hormone insulin. Normally insulin produced when glucose enters the circulation after a meal triggers glucose uptake into cells. Under conditions of insulin resistance, the cells in the body are resistant to the insulin produced after a meal, preventing glucose uptake and leading to high blood sugar.

[0864] The kynurenine hypothesis of diabetes is based on evidence of diabetogenic effects of the kynurenine metabolite Xanthurenic Acid (XA) and the realization that the KP is upregulated by low-grade inflammation and stress, two conditions involved in the pathogenesis of insulin resistance, and of diabetes type I and diabetes type II. Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, possibly due to chronic stress or the low-grade inflammation, which are risk factors for T2DM. As such, the production of kynurenine metabolites can function as a regulatory mechanism to attenuate damage by the inflammation- induced production of reactive oxygen species. [0865] As such inhibition of IDO or decreasing levels of tryptophan and/or kynurenine have been proposed as strategies for the treatment of diabetes. In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of diabetes. In some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic levels of available tryptophan and/or systemic levels of kynurenine and/or other tryptophan metabolite levels.

[0866] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the disorders described herein, e.g., insulin resistance, diabetes, type I and type II. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the disorders described herein, e.g., insulin resistance, diabetes type I and diabetes type II.

[0867] Increasing evidence, both functional and morphological, supports the concept of increased intestinal permeability, altered epithelial barrier function, dysbiosis, changed mucosal innate and adaptive immunity as intrinsic characteristics of type ldiabetes(TlD) in both humans and animal models of the disease (see, e.g., Li and Atkinson, Pediatric Diabetes 2015: 16: 485-492; The role for gut permeability in the pathogenesis of type 1 diabetes - a solid or leaky concept?). In a study with diabetes patients with islet autoimmunity an increase in intestinal permeability to the disaccharide lactulose, indicative of a damaged barrier, relative to controls was observed (Bosi E, et al., Increased intestinal permeability precedes clinical onset of type 1 diabetes. Diabetologia 2006: 49: 2824-2827).

[0868] T2DM is also a disease characterized by a component of intestinal dysfunction. The gut microbiota is altered in type 2 diabetes. Disturbance of intestinal homeostasis, e.g., throught the effects of a western style obesogenic diet, then leads to excessive bacterial fragments/products internal diffusion, which promotes inflammation in key insulin-responsive tissues, resulting in insulin resistance. In contrast, Intake of diets rich in tryptophan may have a positive impact on insulin sensitivity through microbial metabolism of tryptophan to the indole-3-propionic acid, which can be a potential drug target for management of insulin resistance (Khan et al., Microbial Modulation of Insulin Sensitivity; cell Metabolism, Volume 20, Issue 5, 4 November 2014, Pages 753-760).

[0869] Therefore, therapeutic interventions aimed at the gut-pancreas axis, such as improving gut barrier function and the reduction of pro-inflammatory events, have been proposed for prevention, management, and/or treatment of both type 1 and type 2 diabetes and may prove promising for future therapeutic interventions. In some embodiments, the genetically engineered bacteria improve gut barrier function and decrease the "leaky gut" syndrome.

[0870] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of type 1, type 2 diabetes and/or insulin resistance, comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some

embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

[0871] In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

[0872] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of type 2 diabetes (T2DM). In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0873] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein.

[0874] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein., in the subject, e.g., in the serum and/or in the gut.

[0875] [0178] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream

kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein.

[0876] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0877] [0180] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and elsewhere herein. [0878] In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some

embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of T2DM. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

[0879] In some embodiments, the genetically engineered bacteria produce IL- 22, e.g., for the treatment of diabetes and other metabolic disease described herein.In certain embodiments, one or more of these circuits may be combined for the treatment of type 2 diabetes. In a non- limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of type 2 diabetes.

Metabolic Syndrome and Obesity

[0880] Metabolic Syndrome affects approximately 20-30% of the middle-aged population, and represents an increased risk to cardiovascular disorders, the leading cause of death in the United States. Obesity, dyslipidemia, hypertension, and type 2 diabetes are described as metabolic syndrome. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of metabolic syndrome and /or obesity.

[0881] Obesity is a common, deadly, and costly disease in developed countries which impacts all age groups, race, and gender. Obesity can be classified as an inflammatory disease because it is associated with immune activation and a chronic, low-grade systemic inflammation. The basic impetus for obesity is over nutrition and a lack of physical exercise. Over nutrition leads to an excess intake of tryptophan (TRP)— an essential amino acid, a precursor for serotonin (5-HT) and melatonin, and a key player in the caloric intake regulation. Yet, the circulating levels of TRP have been shown to be low in morbidly obese subjects (Brandacher G, Winkler C, Aigner F, et al. Bariatric surgery cannot prevent tryptophan depletion due to chronic immune activation in morbidly obese subjects. Obes Surg 2006;16:541-548).

[0882] An upregulation of IDO activity, essentially caused by chronic immune- mediated inflammation, could therefore be a key component in the initiation and propagation of obesity and the associated metabolic syndrome, by causing decreased TRP availability for the methoxyindole pathway. Serotonin regulates carbohydrate and fat intake, relieves stress which is another caloric intake trigger, and inhibits neuropeptide Y (NYP)— one of the most potent orexigenic peptides in the

hypothalamus. In connections with sleep, melatonin also plays a critical role in caloric intake regulation. Sleep duration has been inversely linked to leptin levels and food consumption. Sleep deprivation upregulates orexin activity, which then activates NYP and induces hunger. As such, increasing levels of serotonin and/or melatonin production is one strategy in th treatment of such disorders. Thus, in some embodiments, the genetically engineered bacteria produce one or more indole metabolites, which in turn can influence host serotonin production. In other embodments, the genetically engineered bacteria comprise circuitry which modulates the systemic availability of tryptophan and one or more of its metabolites.

[0883] Insulin resistance and T2DM are characterized by low-grade

inflammation. In this respect, LPS trigger a low-grade inflammatory response, and the process of endotoxemia can therefore result in the development of insulin resistance and other metabolic disorders. Several of the metabolites produced by the genetically engineered bacteria described herein are useful in the reduction of inflammation. For example, butyrate, contributes to maintaining intestinal integrity. Other antiinflammatory metabolites as described herein may also be useful in the treatment of type 2 diabetes. Thus, in some embodiments, the genetically engineered bacteria comprise circuitry for the production of ant i- inflammatory molecules, e.g., Ahr agonists described herein. In some embodiments, the genetically engineered bacteria produce one or more anti- inflammatory molecules and one or more gut barrier enhancer molecules.

[0884] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the metabolic syndrome and obesity. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of metabolic syndrome and obesity.

[0885] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the subject, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B, and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0886] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, and elsewhere herein, including but not limited to, Tryptamine, Indole-3- acetaldehyde, Indole-3-acetic acid, indole-3- propionic acid, Indole, 6-formylindolo(3,2- b)carbazole, Kynurenic acid, Indole-3-aldehyde; 3,3'-Diindolylmethane. . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table Table 23, FIG. 6A and FIG 6B and elsewhere herein.

[0887] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the subject, e.g., in the serum and/or in the gut e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0888] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B, and elsewhere herein.

[0889] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the subject, e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of obesity. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0890] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and FIG 6B and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and FIG. 2 and elsewhere herein, e.g., for the prevention, treatment, and/or management of obesity.

[0891] In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some

embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

[0892] In certain embodiments, one or more of these circuits may be combined for the treatment of obesity. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of obesity. Further combinations may include cytokine producing circuits, such as IL-22.

[0893] In one embodiment the genetically engineered bacteria comprise gene sequences encoding one or more enzymes for the production of serotonin from tryptophan. In one embodiment the genetically engineered bacteria trigger the host cells to release serotonin through the production of indole metabolites. In some

embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding enzymes for the production of one or more indole metabolites and/or for the production of tryptophan.

[0894] In some embodiments, the genetically engineered bacteria promote gut barrier health and treat or prevent inflammation, e.g., through the production of Ahr agonists. In some embodiments, the genetically engineered bacteria can produce tryptophan and re-plenish low tryptophan levels in obese individual.

Nonalcoholic Steatohepatitis (NASH)

[0895] Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases. Nonalcoholic fatty liver disease is a component of metabolic syndrome and a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Simple liver steatosis is defined as a benign form of NAFLD with minimal risk of progression, in contrast to NASH, which tends to progress to cirrhosis in up to 20% of patients and can subsequently lead to liver failure or hepatocellular carcinoma. Hepatic steatosis occurs when the amount of imported and synthesized lipids exceeds the export or catabolism in hepatocytes. An excess intake of fat or carbohydrate is the main cause of hepatic steatosis. NAFLD patients exhibit signs of liver inflammation and increased hepatic lipid accumulation. In addition, the development of NAFLD in obese individuals is closely associated with insulin resistance and other metabolic disorders and thus might be of clinical relevance). L- tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of NAFLD and/or NASH.

[0896] The development of NASH has been considered a "two hit" process. Insulin resistance (with increased lipogenesis), found in obesity and type II diabetes, has been considered the most important factor in the development of hepatic steatosis (the "first hit"). The "second hit" results in necroinflammatory activity and fibrosis. Because the "second hit can be caused by a number of different mechanisms, including increased gut permeability, and resulting gut-derived endotoxins (e.g., LPS) in the liver, mitochondrial dysfunction, oxidative stress, and/or or proinflammatory cytokines (TNF- alpha, interleukins, e.g., IL-1JL-6, and IL-8), more recently, a concept of multiple hits has been developed. Moreover, inflammation can occasionally precede steatosis and patients with NASH can present without much steatosis, suggesting that inflammation can occur first, indicating that many of these hits can occur in different orders or in parallel at the same time. Contributing factors to these hits include intestinal dysbiosis, dietary factors, changes to intestinal permeability, as well as endoplasmic reticulum stress and activation of additional signalling pathways. Thus, in some embodiments, the genetically engineered bacteria comprise one or more genetic circuits for the production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules, e.g., Ahr agonists, described herein.

[0897] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the metabolic disorders described herein, e.g., NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the metabolic disorders described herein, e.g., NAFLD and/or NASH.

[0898] In some embodiments, the genetically engineered bacteria are capable of treating, preventing or managing NASH. In some embodiments, the genetically engineered bacteria comprise a circuit for melatonin production. In some embodiments, the bacteria comprise a circuit for kynurenine production and or tryptophan degradation. In some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites. In some embodiments, the bacteria can reduce or increase systemic levels of tryptophan available for serotonin production. In some embodiments, the bacteria comprise a circuit of serotonin catabolism. In some embodiments, the genetically engineered bacteria express MAO. In some embodiments, the circuits are under control of inflammatory stimuli. In some embodiments, the circuits are induced in low oxygen conditions.

[0899] Evidence is increasing that the gut and liver have multiple levels of associated interdependence, and disturbance of the gut-liver axis has been implicated in a number of conditions linked to obesity, including NAFLD and NASH. The liver, which receives 70% of its blood supply from the gut through the portal venous system, is significantly affected by the gut and its contents. In NASH the liver is exposed to potentially harmful substances derived from the gut (thought increased gut permeability and reduced intestinal integrity), including translocated bacteria, LPS and endotoxins, food antigens, as well as secreted cytokines. Tight junction proteins, such as zonula occludens, normally seal the junction between intestinal endothelial cells at their apical aspect and thus have a vital role in preventing translocation of harmful substances from the gut into the portal system. In NAFLD/NASH, these tight junctions are disrupted, increasing mucosal permeability and exposing both the gut mucosal cells and the liver to potentially pro-inflammatory bacterial products. Translocated microbial products might contribute to the pathogenesis of fatty liver disease by several mechanisms, including stimulating pro-inflammatory and profibrotic pathways via a range of cytokines.

[0900] As such, one strategy in the treatment, prevention, and/or management of NASH may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of ant i- inflammatory effectors, e.g. Ahr agonists described herein. Any such circuits may be combined with a SCFA production circuit.

[0901] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the metabolic disorders described herein, e.g., NASH. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the metabolic disorders described herein, e.g., NASH.

[0902] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream indole tryptophan metabolites described herein, including, but not limited to those listed in Fig. 6A and Fig. 6B and Table 23, and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0903] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more indole tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32.

[0904] In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan

metabolites, including, but not limited to those listed in Fig. 6A and Fig. 6B and Table 23, and elsewhere herein, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Fig. 6A and Fig. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Fig. 6A and Fig. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Fig. 6A and Fig. 6B and Table 23, and elsewhere herein.

[0905] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Fig. 6A and Fig. 6B and Table 23, and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0906] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein. In some

embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream

kynurenine metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in FIG. 6A and FIG. 6B and Table 23, and elsewhere herein., e.g., for the treatment, prevention and/or management of NASH

[0907] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the subject, e.g., in the serum and/or in the gut e.g., for the treatment, prevention and/or management of NASH. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B and elsewhere herein, in the subject, e.g., in the serum and/or in the gut, e.g., for the treatment, prevention and/or management of NASH.

[0908] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 32. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan

metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13, FIG. 34, FIG. 35A, FIG. 35B, and elsewhere herein, e.g., for the treatment, prevention and/or management of NASH. [0909] In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some

embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the treatment, prevention and/or management of NASH. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios, e.g., for the prevention, management and/or treatment of NASH.

Hepatic Encephalopathy

[0910] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of HE. Hepatic encephalopathy (HE) is a syndrome observed in patients with cirrhosis. Hepatic encephalopathy is defined as a spectrum of neuropsychiatric abnormalities in patients with liver dysfunction. Hepatic encephalopathy is characterized by personality changes, intellectual impairment, and a depressed level of consciousness. The pathogenesis of HE is thought to be related to high ammonia levels as a result of liver failure and/or due to the presence of porto-systemic shunts in patients with cirrhosisAdditionally, inflammation and oxidative and nitrosative stress play a role in the development of HE.

[0911] An increased amount of tryptophan in plasma, cerebrospinal fluid, or brain of patients affected by hepatic encephalopathy and animal models of hepatic encephalopathy had been observed as early as the 1980s. High levels of QUIN are observed in rat models of HE, which is likely involved in the pathogenesis of this disorder (Moroni, F., et al. "Content of QUIN and of other tryptophan metabolites increases in brain regions of rats used as experimental models of hepatic

encephalopathy." Journal of neurochemistry 46.3 (1986): 869-874). QUIN is also increased systemically in serum in hepatic encephalopathy patients with severe liver dysfunction, indicating that QUIN may play a role in the induction of neurological dysfunction, neuropathy and encephalopathy in the disease (Imad Lahdou et al., Increased serum levels of QUIN indicate enhanced severity of hepatic dysfunction in patients with liver cirrhosis Human Immunology 74 (2013) 60-66). Thus, in some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of HE comprise comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites, i.e. can limit the availability of TRP in the brain.

[0912] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the disorders described herein, e.g., HE. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the disorders described herein, e.g., HE.

[0913] There is evidence that HE is linked to alterations in gut microbiota and their by-products such as amino acid metabolites (indoles, oxindoles), endotoxins, etc. These factors, superimposed on a background of leaky intestinal barrier and immune dysfunction, are involved in the pathogenesis of HE (gut-liver axis). Gut and liver share a close relationship. The liver, which receives 70% of its blood supply from the gut through the portal venous system, is significantly affected by the gut and its contents.

[0914] In cirrhosis, changes in intestinal tight junctional proteins have been described; though the pathophysiology is not clear, alcohol metabolites and

proinflammatory cytokines have been postulated to result in leaky intestine (Quigley E.M., Stanton C, Murphy E.F. The gut microbiota and the liver. Pathophysiological and clinical implications. J Hepatol.2013;58: 1020-1027). Intestinal barrier dysfunction and systemic inflammation, altered gut flora and their by-products play an important role in the pathogenesis of HE. Impaired intestinal barrier integrity, results in increased bacterial translocation and release of endotoxins (lipopolysaccharides, flagellin, peptidoglycan, and microbial nucleic acids) in circulation, and systemic inflammation. Thus, in some embodiments, the genetically engineered bacteria comprise one or more genetic circuits for the production of one or more gut barrier enhancer molecules and/or anit- inflammatory molecules, e.g., Ahr agonists, described herein, e.g., for the treatment, prevention, and/or management of HE.

[0915] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of HE. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of HE.

Prader Willi Syndrome

[0916] Prader-Willi syndrome (OMIM 176270) is a complex genetic neurodevelopmental disorder with manifested early in failure to thrive, feeding difficulties during infancy, hypogonadism/hypogenitalism, growth hormone deficiency, and typically a paternal 15ql l-ql3 chromosome deletion. In early childhood trough alduhood, food seeking behaviors and hyperphagia are noted along with a low metabolic rate and decreased physical activity leading to obesity which can be life- threatening, if not controlled. PWS is considered the most common syndromic cause of life threatening obesity in childhood (Buttler et al., Am J Med Genet A. 2015

Mar;167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). It has been reported that, when matched for body mass index (BMI), PWS adults had the same prevalence of metabolic syndrome (41.4%) and insulin resistance index as obese controls.

[0917] Prader-Willi syndrome (PWS) has no cure. PWS syndrome individuals present with obesity with hyperphagia and deficit of satiety, and in some cases insulin resistance, that persists thoughout youth and adulthood and remains a critical problem in PWS teenagers and adults because it leads to severe complications, such as limb edema, cardiac or respiratory failure, and physical disabilities. Severe obesity, and food seeking therfroe remains the larges problem with PWS. Access to food must be strictly supervised and limited. Therefore, agents which modulate satiety and orh insulin levels may be useful in the treatment of PWS.

[0918] In additiona, increased inflammatory markers and cytokine levels in the plasma have been observed in PWS individuals. These cytokines serve as chemoattractants for recruitment of immune cells and indicate an inflammatory component in PWS, which underlies certain aspects of the pathology (Buttler et al.„ Am J Med Genet A. 2015 Mar;167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). Therefore, ant i- inflammatory agents may be useful in the treatment of certain aspects of PWS.

[0919] Thus, in some embodiments, the genetically engineered bacteria comprise one or more genetic circuits for the production of one or anti- inflammatory molecules, e.g., Ahr agonists, described herein. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety. In some embodiments, such circuits are combined with circuits for the production of gut barrier function enhancers, e.g., SCFA, e.g., butyrate and/or satiety effectors, e.g., GLP1.

[0920] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the subjecte.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the subjecte.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the subjecte.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, in the subjecte.g., in the serum and/or in the gut.

[0921] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the subjecte.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the subject e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the subject e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the subject e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, in the subject e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS.

[0922] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the subject e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

[0923] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the subject e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the subject e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the subject e.g., in the serum and/or in the gut. In certain

embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, in the subject e.g., in the serum and/or in the gut, e.g., for the prevention, treatment, and/or management of PWS.

[0924] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the subject e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan

metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, treatment, and/or management of PWS.

[0925] In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some

embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels, e.g., for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

[0926] In certain embodiments, one or more of these circuits may be combined for the treatment of PWS. In a non-limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of PWS.

Cardiovascular Disease

[0927] Cardiovascular disease includes coronary artery diseases (CAD) such as angina and myocardial infarction, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis, caused inter alia by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, and the like. The detection, prevention, and treatment of the underlying risk factors of the metabolic syndrome are a critical approach to lower the cardiovascular disease incidence in the general population.

[0928] Cellular adhesion molecules and oxidative stress play a role in the pathogenesis of atherosclerosis in patients with chronic kidney disease (CKD) and uremia. Uremia is condition that occurs when the kidneys no longer filter properly, and is likely to occur s in the final stage of chronic kidney disease. Several studies in CKD patients have shown that tryptophan metabolites along the kynurenine pathway are increased, possibly as consequence of inflammation. Therefore, anti- inflammatory agents may be useful in the treatment of cardiovascular disease, including CKD and artherosclerosis.Thus, in some embodiments, the genetically engineered bacteria systemically modulate the levels of one or more of tryptophan or onoe or more antiinflammatory metabolites, e.g., Ahr agonists described herein.

[0929] Ischemic stroke, which results from cerebral arterial occlusion, is becoming a major cause of morbidity and mortality in today's society and affects millions of people every year. Currently, the only approved treatment for the acute phase of stroke is the recombinant thrombolytic tissue-type plasminogen activator. Identifying molecules that contribute to the ischemic damage may help to elucidate potential therapeutic targets. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of ischemia and stroke. Inflammation and oxidative stress are also involved in brain damage following stroke, and tryptophan oxidation along the kynurenine pathway contributes to the modulation of oxidative stress. [0930] In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis, ischemic stroke, and/or chronic kidney disease. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments, the circuits stimulate insulin secretion and/or promote satiety.

[0931] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain

embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0932] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan

metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein.

[0933] [0231] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, in the subject, e.g., in the serum and/or in the gut.

[0934] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease.

[0935] [0233] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the subject, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, in the subject, e.g., in the serum and/or in the gut, e.g., for the prevention, management and/or treatment of cardiovascular disease.

[0936] [0234] In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the subject, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in FIG. 2. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 23, FIG. 6A and 6B, and FIG. 2 and elsewhere herein, e.g., for the prevention, management and/or treatment of cardiovascular disease.

[0937] [0235] In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.

[0938] [0236] In certain embodiments, one or more of these circuits may be combined for the treatment of cardionvascular disorders. In a no n- limiting example, SCFA (e.g., butyrate) producing, GLP-1 secreting, and tryptophan pathway modulating (e.g., tryptophan and/or indole metabolite and or/tryptamine producing) cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of cardionvascular disorders. Vascular Biology

Arthero sclerosis

[0939] An impaired intestinal barrier function, resulting bacterial translocation and presence of bacterial products in the circulation can contribute to atherosclerosis and chronic heart failure (CHF) (Rogler and Rosano, Eur Heart J. 2014 Feb;35(7):426- 30. The heart and the gut). Thus, in some embodiments, the genetically engineered bacteria comprise one or more genetic circuits for the production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules, e.g., Ahr agonists, described herein, e.g., for the treatment, prevention, and/or management of chronic heart failure.

[0940] Moreover, kynurenine has been found to participate in several aspects of vascular biology, it has been postulated that KP could be involved in the pathogenesis of atherosclerosis (Niinisalo P, et al. Activation of indoleamine 2,3-dioxygenase- induced tryptophan degradation in advanced atherosclerotic plaques: Tampere vascular study. Annals of medicine. 2010;42:55-63). This is supported by a recent report that in mice the beneficial effect of omega-3 fatty acids on regression of atherosclerotic lesions is mediated largely via IDO induction and kynurenine production in the lesions

(Nakajima K, et al. Orally administered eicosapentaenoic acid induces rapid regression of atherosclerosis via modulating the phenotype of dendritic cells in LDL receptor- deficient mice. Arteriosclerosis, thrombosis, and vascular biology. 2011 ;31: 1963-1972). In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of arthero sclerosis.

[0941] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the cardiovascular disorders described herein. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the cardiovascular disorders described herein. Chronic Kidney Disease

[0942] The terms Chronic kidney disease (CKD) or chronic renal failure (CRF) describe various degrees of decreased renal function, from damaged-at risk through mild, moderate, and severe chronic kidney failure. CKD is associated with an increased risk of cardiovascular disease and chronic renal failure and one of the leading causes of death in the United States. Non-traditional risk factors contributing to cardiovascular pathology in CKD include chronic inflammation, oxidative stress, protein-energy wasting, disordered mineral metabolism, and deficiency of endogenous calcification inhibitors.

In situ

[0943] Several studies in CKD patients have shown that tryptophan metabolites along the kynurenine pathway are increased, possibly as consequence of inflammation (Shefold et al., Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease: a possible link between chronic inflammation and uraemic symptoms Nephrol. Dial. Transplant. (2009) 24 (6): 1901-1908). It has therefore been suggested that the KP activation may be involved in the pathogenesis of CKD, and that KP inhibition may provide an effective strategy to slow down the disease. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of chronic kidney disease. Thus, in some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites.

Gut-renal axis

[0944] Additionally, accumulating evidence over the recent years has suggested the gastrointestinal tract as a major source of chronic inflammation in CKD (see e.g., Lau et al., Nephron. 2015; 130: 92-98; The gut as a source of inflammation in chronic kidney disease). Gut bacterial DNA fragments, endotoxin, and resulting chronic inflammation have been detected in the blood of both pre-dialysis CKD and chronic hemodialysis patients. (Vaziri ND, et 1., Chronic kidney disease causes disruption of gastric and small intestinal epithelial tight junction. Am J Nephrol. 2013;38(2):99-103). Animal studies have confirmed that CKD is associated with depletion of gut epithelial tight junction proteins including occludin and claudin-1 and leaky barrier(Lau et al., Nephron. 2015). Consequently, Thus, in some embodiments, the genetically engineered bacteria comprise one or more genetic circuits for the production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules, e.g., Ahr agonists, described herein, e.g., for the treatment of chronic kidney disease.

[0945] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the vascular disorders described herein, e.g., chronic kidney disease. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the vascular disorders described herein, e.g., chronic kidney disease.

Hypoxia/Ischemia and Sepsis

Intestinal integrity

[0946] Intestinal ischemia/reperfusion (I/R) injury and hypoxic stress are seen in patients with mesenteric artery embolism, traumatic or hemorrhagic shock, and inflammatory bowel disease, and may trigger mucosal barrier damage and enteric bacterial translocation (BT), leading to development of septic complications (see e.g., Lu et al., Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion; Laboratory Investigation (2012) 92, 783-796). Thus, in some embodiments, the genetically engineered bacteria comprise one or more genetic circuits for the production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules, e.g., Ahr agonists, described herein, for the prevention of ischemia.

In situ

[033] Kynurenine produced by endothelial-derived IDO acts as a vascular relaxing factor contributing to vasodilatation in septic shock, and pharmacologic inhibition of IDO improved survival in a mouse septic-shock model (Jung ID, et al. Blockade of indoleamine 2,3-dioxygenase protects mice against lipopolysaccharide- induced endotoxin shock. J Immunol. 2009;182:3146-3154). It is thought that increased production of kynurenine causes immunosuppressive effects and ultimately promotes a hypo inflammatory phase, leading to paralysis of the immune system and endotoxin tolerance. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of sepsis.

[034] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the vascular disorders described herein, e.g., hypoxia, ischemia, or sepsis. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the vascular disorders described herein, e.g., hypoxia, ischemia or sepsis.

Hypertension

[0947] Hypertension is abnormally high blood pressure. Dysbiosis of the gut microbiota has long been associated with hypertension, and reducing the

Firmicutes/Bacteroidetes was showin to have a positive effect on hypertension, indicating that hypertension can be positively influenced from the gut.

[0948] Kynurenine acts as an endothelium-derived relaxing factor that is able to decrease blood pressure in hypertensive rats (Wang et al., Kynurenine is a novel endothelium-derived relaxing factor produced during inflammation; Nat Med. 2010 Mar; 16(3): 279-285). Thus, in some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites. In some embodiments, the genetically engineered bacteria comprise any of the TRP/KP metabolism modulating cassettes described herein, and can beneficically and context-dependently influence immune suppression and blood pressure through changes in the TRP:KYN ratio in this first compartment.

[0949] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the vascular disorders described herein, e.g., hypertension. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the vascular disorders described herein, e.g., hypertension. Neurologic function

[0950] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, correction and/or management of Neurologic function. Mounting evidence suggests that the microbiome has a key role in influencing the development and function of the nervous system through its interaction with the gut-brain axis behaviour (reviewed in Kennedy et al., Kynurenine pathway metabolism and the microbiota-gut-brain axis). Communication between the brain and gut occurs along network of pathways collectively termed the brain-gut axis, and which include the CNS, enteric nervous system (ENS), sympathetic and parasympathetic branches of the autonomic nervous system, neuroendocrine and neuroimmune pathways, and the gut microbiota.

[0951] One key aspect is the microbial regulation of circulating tryptophan availability, which plays a role in (1) the regulation of serotonin synthesis, (2) the regulation of kynurenine pathway metabolism (3) additionally, the production of tryptophan metabolites, such as indoles in the gut, in turn regulate serotonin production and also have inti- inflammatory function in the CNS, through Ahr agonism.

[0952] The term neurodegenerative disease refers to the pathology associated with disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease and Parkinson's disease (Maragakis et al., Mechanisms of Disease: astrocytes in neurodegenerative disease; Nature Clinical Practice Neurology (2006) 2, 679-689). Mouse models of neurodegenerative diseases indicate that astrocyte- specific pathologies contribute to neurodegeneration, and that modulation of astocyte activity is considered a point of therapeutic intervention for these diseases.

[0953] Astrocytes are the most abundant cells in the central nervous system (CNS). Under normal conditions astrocytes modulate synaptic activity, and provide nutrients and support needed for neuronal survival. In the context of neuroinflammation, astrocytes control CNS infiltration by peripheral pro-inflammatory leukocytes and regulate the activity of microglia, oligodendrocytes and cells of the adaptive immune system. Thus, therapeutic regulation of astrocyte activation during CNS inflammation, provides potential targets for a number of neurodegenerative diseases.

[0954] Recent studies have shown that tryptophan metabolites taken in through the diet modulate astrocyte function and have an ant i- inflammatory effect in the CNS, through a mechanism dependent on Ahr agonism (Rothhammer et al., Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor Nat Med. 2016 Jun; 22(6): 586-597).

[0955] In some embodiments, the genetically engineered bacteria modulate astrocyte function. In some embodiments, the genetically engineered bacteria reduce CNS inflammation, e.g., as seen in MS and other CNS disorders. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequences encoding enzymes for the production of Ahr agonists. In some embodiments, genetically engineered bacteria comprising any of the one or more gene sequence(s) for the production of indole metabolites (e.g., tryptamine, indole-acteic acid, indole-3- propionate circuits) described herein are useful for modulating astocyte function and reducing CNS inflammation.

[0956] Kynurenine pathway metabolites have a broad range of functions in the brain and play a key role in a number of neurological disorders. For example, high levels of KYNA are observed in the human brain. Kynurenine, readily enters the brain from the circulation, where levels are controlled by availability of kynurenine and tryptophan, and is taken up by glial cells. The synthesis of 3-HK and further downstream kynurenine pathway metabolites occurs in microglia, whereas KYNA is formed in astrocytes, i.e, the two main arms of the kynurenine pathway are physically segregated in the brain (Guillemin GJ, et al. Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection. J. Neurochem. 2001;78:842-853).

[0957] In contrast to several other kynurenine pathway metabolites, and because of their polar nature and the lack of transport processes, KYNA and QUIN do not cross the blood-brain barrier and must be formed locally within the brain from kynurenine and 3 -HAN A. Fluctuations in the blood levels of tryptophan, kynurenine and 3-HK, which can be influenced by tryptophan levels produced in the gut, directly affect metabolism in the brain kynurenine pathway, including the synthesis of neuroprotective KYNA and neurotoxic QUIN. As such, in some embodiments, the genetically engineered bacteria comprise circuitry which modulates systemic availability of tryptophan and/or one or more of its metabolites, e.g., to modulate neurotoxix and/or neuroprotective tryptophan metabolites in the brain.

[0958] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein.

[0959] In some embodiments, the genetically engineered bacteria comprise circuits, which can reduce QUIN and/or increase KYNA production in the brain, e.g., through systemic control or modulation of tryptophan and/or kynurenine levels. In some embodiments, the genetically engineered bacteria can be combined with one or more additional conventional or other treatment(s) described herein or known in the art for the treatment, prevention and/or management of neurological disorders.

Multiple Sclerosis

[0960] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of multiple sclerosis (MS). MS is a chronic disease with mainly inflammatory features in the beginning and later neurodegenerative processes take over. MS is characterized by breakdown of the blood-brain barrier (BBB) and demyelination of the central nervous system (CNS) due to infiltrating self-reactive T cells recognizing myelin antigens. The inflammation in MS may trigger a pathogenic cascade of events leading to neurodegeneration, amplified by mechanisms related to brain aging and an accumulated disease burden.

[0961] Environmental factors are known to contribute to MS, but the identity of these factors and their mechanisms of action have yet to be further characterized.

Recently, imbalances in the uptake, production and/or degradation of AHR agonists, such as tryptophan metabolites described herein, may have been identified as potential contributors to the pathogenesis of MS and other immune mediated diseases

(Rothhammer et al., Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor Nat Med. 2016 Jun; 22(6): 586-597)). Experimental autoimmune encephalitis (EAE) is a T-cell-mediated autoimmune animal model for MS that is histologically similar to human MS. In this study, disease scores in the experimental encephalitis model (EAE) were increased following antibiotic treatment (which removes the microbiota), and the inflammation was reduced in antibiotic-treated mice supplemented with the tryptophan metabolites indole, indoxyl-3-sulfate, indole-3-propionic acid, indole-3-aldehyde or the bacterial enzyme tryptophanase. Dietary indole metabolites reduced astrocyte inflammatatory markers. Moreover, in MS patients, circulating levels of these and other AHR agonists were decreased.

[0962] As such, in some embodiments, one or more of any of the circuits for the production of one or more indole metabolites described herein and optionally circuits for the production of tryptophan can be used for the prevention, management, or treatment of CNS disorders such as, multiple sclerosis. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production of one or more indole metabolites described herein and optionally circuits for the production of tryptophan described herein and are administered for the prevention, management, or treatment of any of the CNS disorders described herein, e.g., multiple sclerosis.

[035] Additionally, a role for the gut and the gut microbiota in the development of EAE in mice has also been shown (Berer et al., 2011, NATURE, VOL 479: 538-542; Commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelinationFurthermore, Nouri et al. (2014) examined the intestinal tract of mice with EAE and demonstrated an increased gut permeability and an altered mucosal structure including inflammation in the small intestine, with concurrent zonulin upregulation (PLoS One. 2014; 9(9): el06335 Intestinal Barrier Dysfunction Develops at the Onset of Experimental Autoimmune Encephalomyelitis, and Can Be Induced by Adoptive Transfer of Auto-Reactive T Cells). In this model, the leaky gut may act to support disease progression, and as a result represent a potential therapeutic target in MS. Therefore, genetically engineered bacteria which function ot promote gut barrier health, as described herein, can be useful in the treatment of MS. can be targeted by the engineered bacteria of the disclosure.

[036] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of MS comprise one or more gene(s) or gene sequence(s) for the production of kynurenine and/or kynurenic acid and/or indole metabolites described herein for the treatment, management and/or prevention of autoimmune diseases. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene sequence(s) for the conversion tryptophan into kynurenine, kynurenic acid and/or indole metabolites described herein. In some embodiments, the genetically engineered bacteria are capable of taking up tryptophan from the extracellular environment. In some embodiments, the genetically engineered bacteria are capable of producing tryptophan, e.g., through any of the genomic modifications and/or circuits described herein. In some embodiments, the tryptophan produced or taken up from the extracellular space is used for the production of one or more of kynurenine, kynurenic acid and/or indole metabolites described herein.

[037] In the EAE model, Kwidzinski et al. (Kwidzinski Eet al., Indolamine

2,3-dioxygenase is expressed in the CNS and down-regulates autoimmune

inflammation. FASEB J. 2005;19: 1347-1349) demonstrated the inhibition of the IDOl activity significantly decreases the neuroinflammatory process, resulting in a decrease of the exacerbation of the disease, possibly through prevention of the production and accumulation of KP downstream metabolites QUIN and 3-HK (Radja et al., 2015 Int J Mol Sci. 2015 Aug; 16(8): 18270-18282. Kynurenines and Multiple Sclerosis: The Dialogue between the Immune System and the Central Nervous System, and references therein).

[038] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the autoimmune and CNS disorders described herein, e.g., multiple sclerosis. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the autoimmune disorders described herein, e.g., multiple sclerosis.

[039] In some embodiments, the genetically engineered bacteria for the treatment, management and/or prevention of autoimmune diseases comprise one or more gene(s) or gene sequence(s) for the production of kynureninase, e.g., for the conversion of KYN to AA, e.g., from Pseudomonas luminescens, as described herein.

[040]

Huntington's Disease

[0963] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Huntington's disease. Huntington's disease is an inherited disease that causes the progressive breakdown (degeneration) of nerve cells in the brain. Huntington's disease has a broad impact on a person's functional abilities and usually results in movement, thinking (cognitive) and psychiatric disorders. The disease is caused by an expanded CAG trinucleotide repeat (of variable length) in HIT, the gene that encodes the protein huntingtin. Several mechanisms that are not mutually exclusive have been suggested to play a role in the pathogenesis of HD. These mechanisms include

neuroinflammation, transcriptional dysregulatioii, excitotoxicitv, and mitochondrial dysfunction. Damage of mutant Htt expressing neurons is suggested to lead to microglia activation, which includes secretion of cytokines as well as increased IDC) transcription and generation of neuroactive kynurenine metabolites (Schwarcz and Pellicciari, 2002). Indeed, increased levels of metabolites have been reported in human post-mortem brain as well as in various animal models of HD.

[0964] Several lines of evidence suggest both peripheral and central

neuroinflammation occurs in HD. Activation of the peripheral immune system is for example indicated by elevation in several plasma cytokines in HD patients including IL- 6, IL-8, IL-4, IL- 10, and TNF-a (Campbell et al., 2014 Kynurenines in CNS disease: regulation by inflammatory cytokines).

[0965] Studies in post-mortem brains revealed that QUIN levels are

substantially elevated in the initial stages of the disease, and especially in those brain regions (neostriatum, cortex) that suffer the most damage. This increase, which coincides with early activation of microglia, generates QUIN concentrations that are clearly capable of producing excitotoxic neuronal damage.

[0966] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., Huntington's disease. In some

embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., Huntington's disease. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes for the production of one or more anti- inflammatory immune modulators, e.g., prevent, manage or treat neuro inflammation in Huntingtons disease. Non- limiting examples of such modulators include one or more Ahr agonists, such as one or more indole metabolites described herein, or kynurenine or kynurenic acid. In some embodiments, genetically engineered bacteria comprise gene sequences for the expression of enzymes, which systemically prevent the accumulation of toxic KP metabolites and promote the production of neuroprotective metabolites, e.g., KYNA.

Parkinson's Disease

[0967] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Parkinsons' s disease. Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder characterized by loss of dopaminergic neurons in the midbrain and presence of protein inclusions called Lewy bodies (Zinger et al., 2011). The detailed pathogenesis of PD is not known, but several mechanisms have been proposed including mitochondrial dysfunction, neurotoxicity from excessive glutamatergic activity, and reactive oxygen species. Neuro inflammation, as measured by the presence of activated microglia in PD brain, as well as excessive production of cytokines and dysregulation of the KP have been suggested to be involved in these complex pathogenic events.

[0968] Changes in kynurenine metabolism have been reported in post-mortem PD brain and mouse models of PD. In the basal ganglia of patients with Parkinson's disease, the concentration of 3-HK is increased, whereas kynurenine and KYNA levels are slightly reduced (Ogawa T, et al. Kynurenine pathway abnormalities in Parkinson's disease. Neurology. 1992;42: 1702-1706.)

[0969] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., Parkinson's disease. In some

embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., Parkinson's disease.

[0970] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes for the production of one or more anti- inflammatory immune modulators, e.g., prevent, manage or treat neuro inflammation in Parkinsons disease. Non- limiting examples of such modulators include one or more Ahr agonists, such as one or more indole metabolites described herein, or kynurenine or kynurenic acid. In some embodiments, genetically engineered bacteria express circuits described above, which prevent the accumulation of toxic KP metabolites and produce

neuroprotective metabolites, e.g., KYNA. In some embodiments, the genetically engineered bacteria comprise circuits, which allow a change in QIUN: KYNA ratio and for an overall reduction of QUIN and other neurotoxic metabolites, as described above. Alzheimers Disease

[0971] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Alzheimers' s disease. Alzheimer's disease (AD) is a progressive neurological disorder characterized by impaired memory, cognitive decline, and dementia. Currently there is still only a limited understanding of AD etiology, particularly in late onset AD. AD pathology hallmarks are the presence of β-amyloid (Αβ) plaques, neurofibrillary tangles, and gliosis. Multiple hypotheses exist regarding factors that contribute to the development and progression of AD including substantial evidence for neuroinflammatory processes. Of note, microglia activation states correlate with disease progression and levels of dementia (Cagnin, A., Kassiou, M., Meikle, S. R., and Banati, R. B. (2006). In vivo evidence for microglial activation in neurodegenerative dementia. Acta Neurol. Scand. Suppl. 185, 107-114.). Analysis of serum samples and post-mortem brain tissue from AD patients demonstrate an imbalance in pro- and ant i- inflammatory cytokines, as well as irregular tryptophan metabolism through activation of microglia and astrocytes (Campbell et al., Kynurenines in CNS disease: regulation by inflammatory cytokines Front. Neurosci., 06 February 2014, and references therein).

[0972] Among the neurochemical changes in AD, IFN-γ, TNF-a, IL-Ιβ, IL-2, and IL-8 are elevated along with lower levels of tryptophan and increased kynurenine levels in serum samples from AD patients. It is thought that QUIN-induced

excitotoxicity or oxidative stress might participate in the pathogenic process (Guillemin GJ, Brew BJ, Noonan CE, Takikawa O, Cullen KM. Indoleamine 2,3 dioxygenase and QUIN immunoreactivity in Alzheimer's disease hippocampus. Neuropathol. Appl. Neurobiol.2005;31:395-404). [0973] In addition, indoles (Indole-3-propionic acid) have been found beneficial Alzheimers; IPA completely protected primary neurons and neuroblastoma cells against oxidative damage and death caused by exposure to Abeta, by inhibition of superoxide dismutase, or by treatment with hydrogen peroxide ( Chyan et al., b- Amyloid by an Endogenous Melatonin-related Indole Structure, Indole-3-propionic Acid J bil. Chem 1999). As such, IPA is considered for the treatment of Alzheimers disease (reviewed in Development of Indole-3-Propionic Acid (OXIGON™) for Alzheimer's Disease, Bendheim et al., 2001, the contents of which is hherien incormprated by reference in its entirety).

[0974] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., Alzheimer's disease. In some

embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., Alzheimer's disease.

[0975] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes for the production of one or more anti- inflammatory immune modulators, e.g., prevent, manage or treat neuro inflammation in Alzheimer disease. Non- limiting examples of such modulators include one or more Ahr agonists, such as one or more indole metabolites described herein, or kynurenine or kynurenic acid. In some embodiments, genetically engineered bacteria comprise one or more gene sequence(s) for the production of IPA, to prevent, manage or treat Alzheimers

[0976] In some embodiments, the genetically engineered bacteria comprise circuits, which allow a change in QIUN: KYNA ratio and for an overall reduction of QUIN and other neurotoxic metabolites, as described above.

Amyotrophic lateral sclerosis (ALS)

[0977] Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease of unknown pathogenesis. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of ALS. The kynurenine pathway, activated during neuroinflammation, is emerging as a possible contributory factor in ALS. The KP is the major route for TRP catabolism. The intermediates generated can be either neurotoxic, such as QUIN, or neuroprotective, such as picolinic acid (PIC), an important endogenous chelator. As the cause of ALS is still unknown, there is presently no efficient treatment for it. Currently, Riluzole is the drug of choice but its effect is relatively modest. Targeting the KP, hence, could offer a new therapeutic option to improve ALS treatment.

[0978] Significantly increased levels of TRP, KYN and QUIN and decreased levels of serum PIC were found in ALS samples, indicating the presence of

neuroinflammation in ALS and providing evidence for the involvement of the KP in ALS. (The kynurenine pathway and inflammation in amyotrophic lateral sclerosis. Chen Y, Stankovic R, Cullen KM, Meininger V, Garner B, Coggan S, Grant R, Brew BJ, Guillemin GJ. Neurotox Res. 2010 Aug;18(2): 132-42).

[0979] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., ALS. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., ALS.

[0980] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes for the production of one or more anti- inflammatory immune modulators, e.g., prevent, manage or treat neuroinflammation in ALS disease. Non- limiting examples of such modulators include one or more Ahr agonists, such as one or more indole metabolites described herein, or kynurenine or kynurenic acid.

[0981] In some embodiments, the genetically engineered bacteria comprise circuits, which allow a change in QIUN: KYNA ratio and for an overall reduction of QUIN and other neurotoxic metabolites, as described above. In other embodiments, the genetically engineered bacteria express one or more gene sequences described herein for the production of tryptophn metabolits.

Seizure Disorders

[041] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of Seizure disorders. It is throught that the pro-convulsant activity of QUIN may at least exacerbate or even cause neuronal hyperactivity and/or excitotoxicity (Lehrmann et ah , 2008).

Furthermore, both QUIN and 3-HK may contribute to neuronal degeneration, further enhancing neuro inflammatory responses at the root of disease pathology.

[042] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., seizure disorders. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., seizure disorders.

[043] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes for the production of one or more antiinflammatory immune modulators, e.g., prevent, manage or treat neuroinflammation in seizure disorders. Non-limiting examples of such modulators include one or more Ahr agonists, such as one or more indole metabolites described herein, or kynurenine or kynurenic acid.

[044] In some embodiments, the genetically engineered bacteria comprise circuits, which allow a change in QIUN: KYNA ratio and for an overall reduction of QUIN and other neurotoxic metabolites, as described above.

[045] In some embodiments, genetically engineered bacteria express circuitry which systemically modulates neuroinflammation, e.g. for the treatment, management and/or prevention of seizure disorders. In some embodiments, the genetically engineered bacteria comprise gene sequences encoding enzymes for the production of Ahr agonists, e.g., as seen in FIG. 6, Table 23, and/or certain kynurenine metabolites.

Major Depressive Disorders

[046] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of depression.

Depression is the most prevalent neuropsychological disorder. Worldwide figures estimate that -20% of people will experience a major depressive episode throughout the course of their lifetime (Kessler et al, 2005). Emerging data show that dysregulation of the immune system, over expression of proinflammatory cytokines, and aberrant tryptophan metabolism are contributing factors at least in a subset of MDD cases.

[047] The kynurenine pathway dysfunction is now increasingly recognized as a major player in the development and symptomatology of depressive disorders; the KYN:TRP ratio in blood is significantly enhanced in patients with depression and correlates with anxiety and cognitive deficits, but not with neurovegetative or somatic symptoms. Systemic IDO activation leading to this increased circulating kynurenine levels, may also lead to depressive behaviors, e.g., enhanced kynurenine influx from the periphery may therefore enhance ratio between the neurotoxic metabolites, i.e. 3-HK and QUIN, and the neuroprotective KYNA (Hanson ND, Owens MJ, Nemeroff CB. Depression, antidepressants, and neurogenesis: a critical reappraisal.

Neuropsychopharmacology. 2011;36:2589-2602; 133. Laugeray A, et al. Peripheral and cerebral metabolic abnormalities of the tryptophan-kynurenine pathway in a murine model of major depression. Behav. Brain Res. 2010).

[048] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., depressive disorders. In some

embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., depressive disorders.

[049] In some embodiments, the genetically engineered bacteria comprise circuits, as described herein, which are useful in modulating the systemic tryptophan and kynurenine levels. In some embodiments, the genetically engineered bacteria comprise circuits, which allow a change in QIUN: KYNA, e.g., an overall reduction of QUIN.

[050] In some embodiments, genetically engineered bacteria express circuitry which systemically modulates neuroinflammation, e.g. for the treatment, management and/or prevention of depressive disorders. In some embodiments, the genetically engineered bacteria comprise gene sequences encoding enzymes for the production of Ahr agonists, e.g., as seen in FIG. 6, Table 23, and/or certain kynurenine metabolites.

Chronic inflammatory syndrome, pain, and depression

[051] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of chronic

inflammatory syndrome, pain, and depression. Chronic inflammatory syndromes are frequently accompanied by comorbidities of heightened pain (nociception) and affective depression. IDO activity in the CNS is elevated in such syndromes and elevated plasma kynurenine has and effect in patients with chronic pain or depression (Dantzer R, et al. From inflammation to sickness and depression: when the immune system subjugates the brain. Nature reviews Neuroscience. 2008;9:46-56). In mice, genetic IDOl ablation or pharmacologic IDO inhibition eliminated behavioral changes linked to 'sickness- induced' depression in a model of chronic mycobacterial infection (O'Connor JC, et al. Induction of IDO by bacille Calmette-Guerin is responsible for development of murine depressive-like behavior. J Immunol. 2009; 182:3202-3212.). Effects of elevated kynurenine may be caused by reduced serotonin levels (due to depletion of TRP, the substrate for serotonin synthesis) and/or release of neurotoxic TRP catabolites.

[052] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., chronic inflammatory syndrome, pain, or depression. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., chronic inflammatory syndrome, or pain, or depression.

[053] In some embodiments, genetically engineered bacteria express circuits described above which can modulate systemic tryptophan and/or kynurenine levels, and which can reduced kynurenine available for conversion to toxic KP metabolites. In other embodiments, the genetically engineered bacteria comprise gene sequences encoding enzymes which produce neuroprotective metabolites, e.g., KYNA. [054] In some embodiments, genetically engineered bacteria express circuitry which systemically modulates neuroinflammation, e.g. for the treatment, management and/or prevention of chronic pain. In some embodiments, the genetically engineered bacteria comprise gene sequences encoding enzymes for the production of Ahr agonists, e.g., as seen in FIG. 6, Table 23, and/or certain kynurenine metabolites.

[055] In other embodiments, the genetically engineered bacteria express endogenus or exogenous circuits for serotonin synthesis as described herein.

Schizophrenia

[056] Schizophrenia is a complex neuropsychiatric disorder affecting approximately 1% of the world population, characterized by positive (delusions, hallucinations, thought disorder), negative (anhedonia, alogia, asociality) and cognitive (deficits in attention, executive function, and memory) symptom clusters, attributed to disturbances in dopaminergic, glutamatergic, and GABAergic neurotransmission (Harrison and Weinberger, 2005; Lewis et ah, 2005). In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of schizophrenia.

[057] Mounting evidence suggests the involvement of KYNA in

schizophrenia. Supporting this possibility, elevated KYNA levels have been detected in cerebrospinal fluid (Schwieler et ah, Increased levels of IL-6 in the cerebrospinal fluid of patients with chronic schizophrenia— significance for activation of the kynurenine pathway; J Psychiatry Neurosci. 2015 Mar; 40(2): 126-133) of schizophrenic patients compared to controls. In rats, acute KYNA elevation in the prefrontal cortex causes the characteristic impairment in cognitive flexibility seen in schizophrenia, and

experimentally induced schizophrenia increases in KYNA synthesis in the brain, (Schwarcz et al. Kynurenines in the mammalian brain: when physiology meets pathology Nature Reviews Neuroscience 13, 465-477 (July 2012), and references therein).

[058] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., schizophrenia. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., schizophrenia.

[059] In some embodiments, genetically engineered bacteria express circuits, which prevent the synthesis of KYNA. In some embodiments, genetically engineered bacteria express circuits which modulate systemic levels of tryptophan and kynurenine, e.g., reduce systemic levels of tryptophan and kynurenine. In some embodiments, genetically engineered bacteria express circuitry which systemically modulates neuroinflammation, e.g. for the treatment, management and/or prevention of

schizophrenia. In some embodiments, the genetically engineered bacteria comprise gene sequences encoding enzymes for the production of Ahr agonists, e.g., as seen in FIG. 6, Table 23, and/or certain kynurenine metabolites.

Attention Deficit Disorder

[060] Attention Deficit-Hyperactivity Disorder (ADHD) is the most commonly diagnosed psychiatric disorder in children and adolescents. Though estimates of prevalence vary widely, it is estimated that -6-8% of school aged children suffer from this disorder (Larson et ah, 2011 ; Willcutt, 2012). Patients show striking neuropsychological performance deficits compared to peers within their age-group which tend to diminish in severity over time.

[061] Recent developments in the study of ADHD suggest that patients may possess minor imbalances in their immunological systems, as measured by increased serum levels of IFN-γ and IL- 13, while also having reduced levels of 3-HK though normal levels of kynurenine (Oades et ah , 2010b). The altered levels of

proinflammatory cytokine production and kynurenine metabolism trended toward normalizing in medicated subjects relative to medication naive patients.

[062] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., ADD In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., ADD.

Autism

[063] Autism and Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder of unknown etiology. Mouse models, such as the inbred mouse strain BTBR T+tf/J (BTBR), which incorporates multiple behavioral phenotypes relevant to symptoms of autism, all research into the causes of autism and the evaluation of potential treatments. BTBR display a selectively reduced social approach, low reciprocal social interactions and impaired juvenile play, as compared with

C57BL/6J (B6) controls. Several interesting single nucleotide polymorphisms (SNPs) in the BTBR genetic background were observed, including a nonsynonymous coding region polymorphism in the KMO gene encoding the enzyme that produces KYNA, the glutamate antagonist with neuroprotective actions. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of autism.

[064] Additionally, alterations to the KP in ASD, i.e., increased production of

QUIN (enhances glutamatergic neurotransmission) have been observed, and the presence of inflammation is known to induce KP activation in ASD (Lim et al., Altered kynurenine pathway metabolism in autism: Implication for immune-induced

glutamatergic activity. Autism Res. 2015 Oct 24). These findings suggest that increased QUIN may be lead to abnormal glutamatergic activity associated with ASD

pathogenesis and may support the ide of treating with ant i- inflammatory or KP inhibitory regimens.

[065] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., autism. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., autism. [066] In some embodiments, genetically engineered bacteria express circuits described above, which prevent the accumulation of toxic KP metabolites and produce neuroprotective metabolites, e.g., KYNA.

Bipolar Disorder and Drug abuse

[067] Bipolar disorder (BD) is a long-recognized severe and common psychiatric disorder, with a complex and often diverse range of presentations. BD is a heterogenous disorder that has traditionally, been defined by the recurrences of manic and depressive episodes, and presents with numerous immune-inflammatory and circadian/sleep abnormalities. In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of bipolar disorder or drug abuse.

[068] Patients with bipolar disorder have increased levels of KYNA in their

CSF compared with healthy volunteers (Olsson et al. Elevated levels of kynurenic acid in the cerebrospinal fluid of patients with bipolar disorder, J Psychiatry Neurosci. 2010 May; 35(3): 195-199.) In the rat brain, elevatated KYNA levels activate dopamine neurons. Elevation of brain KYNA in bipolar disorder is thought to influence glutamatergic, cholinergic and dopaminergic neurotransmission in these patients, and modulation of the KP may be considered as a useful treatment strategy

((Pharmacological elevation of endogenous kynurenic acid levels activates nigral dopamine neurons. Erhardt S, Oberg H, Mathe JM, Engberg G Amino Acids. 2001; 20(4):353-62).

[069] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., bipolar disorder and/or drug abuse. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., bipolar disorder and/or drug abuse. [070] In some embodiments, genetically engineered bacteria express circuits, which prevent the synthesis of KYNA. In some embodiments, genetically engineered bacteria modulate levels of systemic tryptophan and/or kynurenine, e.g., to reduce availability to the brain.

Chronic brain injury

[071] In one strudy following patients with f severe brain injury, subjects showed increased and persistent inflammation and oxidative stress. At baseline and following tryptophan depletion, more tryptophan was converted to kynurenine in patients than controls, but less kynurenine was converted into the neuroprotectant, kynurenic acid. This suggests that neuroprotection by kynurenic acid may be inadequate in brain-damaged patients even many years after injury (Mac Kay et ah , Tryptophan metabolism and oxidative stress in patients with chronic brain injury. Eur J Neurol. 2006 Jan;13(l):30-42). In some embodiments, the genetically engineered bacteria described herein are useful in the treatment and/or management of chronic brain injury.

[072] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., chronic brain injury. In some

embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., chronic brain injury.

Other Diseases

Pellagra

[073] Pellagra is a deficiency disease caused by a lack of nicotinic acid or its precursor tryptophan in the diet. It is characterized by dermatitis, diarrhea, and mental disturbance, and is often linked to overdependence on corn as a staple food. It can also occur if the body fails to absorb these nutrients. It may develop after gastrointestinal diseases or with alcoholism, HIV/ AIDS, or anorexia. [074] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the disorders described herein, e.g., pellagra. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the disorders described herein, e.g., pellagra.

Hepatic porphyria

[075] Hepatic porphyrias is a form of porphyria in which the enzyme deficiency occurs in the liver. The most common neurovisceral complaints in acute hepatic porphyrias are abdominal pain, vomiting, constipation, muscle weakness, mental symptoms, limb, head, neck, chest pain, hypertension, tachycardia, convulsion, sensory loss, fever, respiratory paralysis and diarrhea (Sassa, Modern diagnosis and management of the porphyrias, British Journal of Haematology, 135, 281-292).

Decreased heme synthesis in the liver results in decreased activity of hepatic tryptophan pyrrolase (TP), a heme-dependent enzyme, possibly resulting in increased levels of brain tryptophan and increased turnover of serotonin (Litman, D.A. & Correia, M.A. (1985) Elevated brain tryptophan and enhanced 5-hydroxytryptamine turnover in acute hepatic heme deficiency: clinical implications. Journal of Pharmacology and

Experimental Therpeutics, 232, 337-345).

[076] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., hepatic porphyria. In some embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., hepatic porphyria. Bone morphogenesis

[077] In some embodiments, the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of bone diseases, including but not limited to osteoporosis. Bone remodeling is a process that occurs throughout a person's entire life. Osteoblasts, necessary for this process, are responsible for forming new bone tissue while osteoclasts take part in bone resorption. This complex process is regulated by many factors, some of which are still not well understood. An imbalance between osteoblast and osteoclast action leads to many metabolic diseases, of which osteoporosis is most well-known. The cause of osteoporosis is a imbalance between excessive bone resorption and insufficient bone formation, which leads to increased risk of fractures and significant reduction in quality of life.

[078] Serotonin can exert different effects on bones, which depend on site of serotonin synthesis. Gut-derived serotonin inhibits bone formation (Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis Yadav VK, Balaji S, Suresh PS Nat Med, 2010), whereas brain-derived serotonin enhances bone formation and decreases bone resorption. Melatonin increased differentiation of human mesenchymal stem cells into the osteoblastic cell lineage, and melatonin action on bone results in anabolic and antiresorptive effects (Sanchez-Barcelo et ah, Scientific basis for the potential use of melatonin in bone diseases: osteoporosis and adolescent idiopathic scoliosis; J Osteoporos. 2010 Jun 1 ;2010:830231)

[079] Kynurenine pathway metabolism in patients with osteoporosis, indicating that tryptophan metabolism is altered in osteoporosis in a manner that could contribute to the oxidative stress and, thus, to progress of the disease (reviewed in Michalowska et al, New insights into tryptophan and its metabolites in the regulation of bone metabolism. J Physiol Pharmacol. 2015 Dec;66(6):779-91).

[080] In some embodiments, one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein can be used for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., bone morphogenesis. In some

embodiments, the genetically engineered bacteria comprise one or more of any of the circuits for the production or catabolism of tryptophan and/or one or more of its metabolites described herein and are administered for the prevention, management, or treatment of any of the neurological disorders described herein, e.g., bone

morphogenesis.

Multiple Mechanisms of Action

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

[082] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g. , circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene encoding a particular payload inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene encoding a particular payload inserted at three different insertion sites and three copies of the gene encoding a different payload inserted at three different insertion sites.

[083] In some embodiments, under conditions where the payload is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the payload, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

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

[085] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (Mo As), e.g. , circuits producing multiple copies of the same product or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in Fig. 12. For example, the genetically engineered bacteria may include four copies of kynureninase and/or tyrpophan biosynthesis operon inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ. In another non- limiting example, the genetically engineered bacteria may include four copies of kynurenine biosynthesis enzyme (s) inserted at four different insertion sites, e.g. , malE/K, insB/I, araC/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include three copies of kynureninase and/or tyrpophan biosynthesis operon inserted at three different insertion sites, e.g. , malE/K, insB/I, and lacZ. In another non- limiting example, the genetically engineered bacteria may include three copies of kynurenine biosynthesis enzymes inserted at three different insertion sites, e.g. , malE/K, insB/I, and lacZ. In some embodiments, the genetically engineered bacteria may include one or more circuits for the production or catabolism of tryptophan and/or one of its metabolites (s) inserted at one or more different insertion sites and one or more additional circuits inserted at one or more other insertion sites. In another non-limiting example, the genetically engineered bacteria may include one or more kynurenine degradation enzymes, e.g., kynureninase, inserted at one or more different insertion sites and one or more additional circuits inserted at one or more other insertion sites.

[086] In any of the circuits for production and/or catabolism of tryptophan and/or one of its metabolites can be combined with one or more other, different cicuits for production and/or catabolism of tryptophan and/or one of its metabolites.

[087] In some embodiments, the genetically engineered bacteria comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. A non-limiting example of such a circuit is AtrpR, AtnaA, trpE_fbr (feedback resistant trpE), trpDCBA, aroGfbr (feedback resistant aroG), SerA_fbr (Feedback resistant SerA)). In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the degradation of kynureninase (e.g., kynureninase gene sequences).

[088] In some embodiments, the genetically engineered bacteria comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. A non-limiting example of such a circuit is AtrpR, AtnaA, trpE_fbr (feedback resistant trpE), trpDCBA, aroGfbr (feedback resistant aroG), SerA_fbr (Feedback resistant SerA)). In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of tryptamine. A non-limiting example of such a circuit is AtrpR, AtnaA, trpEfbrDCBA, aroGfbr-tdc (Clostridium sporogenes), SerAfbr.

[089] In some embodiments, the genetically engineered bacteria comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. A non-limiting example of such a circuit is AtrpR, AtnaA, trpE_fbr (feedback resistant trpE), trpDCBA, aroGfbr (feedback resistant aroG), SerA_fbr (Feedback resistant SerA)). In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of indole- 3 -acetic acid. A non-limiting example of such a circuit is : AtrpR, AtnaA, trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iadl.

[090] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of indole- 3 -acetic acid and tryptamine.

[091] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of indole-3-propionic acid.

[092] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of indole-3-propionic acid and tryptamine.

[093] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of indole-3-propionic acid and tryptamine and indole- 3 -acetic acid.

[094] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of one or more indoles shown in FIG. 6 and in Table 23. [095] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the catabolism of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of one or more indoles shown in FIG. 6 and in Table 23.

[096] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the production of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of one or more indoles shown in FIG. 6 and in Table 2.

[097] In some embodiments, the genetically engineered bacteria optionally comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes, for the catabolism of tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more gene sequences, gene cassettes, and/or deletions in endogenous genes for the production of one or more indoles shown in FIG. 6 and in Table 2.

[098] In any of the combination embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the production of a short chain fatty acid.

[099] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the production of a butyrate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional

(combination circuits) described herein further comprise one or more gene sequences for the production of acetate. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of an ant i- inflammatory cytokine. In any of the

embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of IL-10. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of IL-2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of IL-27. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the catabolism of branched chain amino acids.

[0982] In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL- 18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD137 agonist, ICOS agonist, OXO40 agonist, GM-CSF. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion of IL-15. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g. , PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion or display of an anti-PDl antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for production of arginine. In any of the embodiments, described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production and/or catabolism of tryptophan and/or one or more of its metabolites described herein and optionally one or more additional (combination circuits) described herein further comprise one or more gene sequences for the catabolism of adenosine.

[0983] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[0984] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL- 15 and other effectors (IL- 12, IL-2, IL- 15, IL- 18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g. , scFvs, including but not limited to checkpoint inhibitors (e.g. , PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD- 1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[0985] Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication WO2016/201380, the contents of which is herein incorporated by reference in its entirety.

[0986] In any of the embodiments, above and described elsewhere herein, the genetically engineered bacteria may further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art and (7) one or more circuits for the production or degradation of one or more metabolites (e.g., kynurenine, tryptophan, adenosine, arginine) described herein (8) combinations of one or more of such additional circuits.

[0987] In any of the combination embodiments, described above and elsewhere herein, the gene sequences encoding one or more payload combinations are operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is induced under condition(s) associated with immune suppression and/or tumor microenvironment. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with cancer, or certain tissues, immune

suppression, or inflammation, or in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.

[0988] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more payload combinations are are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, the tumor microenvironment or a particular tissue. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table 10-20. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more payload combinations are operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table 21 In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more payload combinations are modified and/or mutated, e.g. , to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.

[0989] In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more payload combinations are be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments, described above and elsewhere herein, the gene sequence encoding one or more payload combinations are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome. Secretion

[0990] In any of the embodiments, described herein, in which the genetically engineered organism, e.g. , engineered bacteria or engineered OV, produces a protein, polypeptide, peptide, or other anti-cancer, gut barrier enhancer, ant i- inflammatory, neuromodulatory,satiety effector, DNA, RNA, small molecule or other molecule intended to be secreted from the microorganism, the engineered microorganism may comprise a secretion mechanism and corresponding gene sequence(s) encoding the secretion system.

[0991] In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting the polypeptide of interest, e.g., a tryptophan metabolism enzyme, such as kynureninase, and others described herein, from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.

[0992] In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a no n- native double membrane- spanning secretion system. Double membrane- spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993 ; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in Fig. 27, Fig. 28, Fig. 29, Fig. 30, Fig. 31, Fig. 32A -32E. Mycobacteria, which have a Gram- negative- like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane- spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane- spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane- spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).

[0993] In some embodiments, in which the one or more proteins of interest or therapeutic proteins are secreted or exported from the microorganism, the engineered microorganism comprises gene sequence(s) that includes a secretion tag. In some embodiments, the one or more proteins of interest or therapeutic proteins include a "secretion tag" of either RNA or peptide origin to direct the one or more proteins of interest or therapeutic proteins to specific secretion systems. For example, a secretion tag for the Type I Hemolysin secretion system is encoded in the C-terminal 53 amino acids of the alpha hemolysin protein (HlyA).

[0994] In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. Fig. 29 shows the alpha-hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. HlyB inserts into inner membrane to form a pore, HlyD aligns HlyB with TolC (outer membrane pore) thereby forming a channel through inner and outer membrane. Natively, this channel is used to secrete HlyA, however, to secrete therapeutic peptide of the present disclosure, the secretion signal- containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide. The C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the one or more proteins of interest or therapeutic proteins into the extracellular milieu. In some embodiments, the one or more proteins of interest or therapeutic proteins contain expressed as fusion protein with the 53 amino acids of the C termini of alpha-hemolysin (hlyA) of E. coli CFT073 (C terminal secretion tag).

[0995] In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. The Type V Auto- secretion System utilizes an N-terminal Sec-dependent peptide tag (inner membrane) and C-terminal tag (outer- membrane). This system uses the Sec-system to get from the cytoplasm to the periplasm. The C-terminal tag then inserts into the outer membrane forming a pore through which the "passenger protein" threads through. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in Fig. 28, a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal, Sec-dependent signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex ('Beta-barrel assembly machinery') where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once across the outer membrane, the passenger is released from the membrane-embedded C-terminal tag by either an autocatalytic, intein-like mechanism (left side of Bam complex) or via a membrane-bound protease (black scissors; right side of Bam complex) (i.e. , OmpT). For example, a membrane-associated peptidase to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.

[0996] The N-terminal tag is removed by the Sec system. Thus, in some embodiments, the secretion system is able to remove this tag before secreting the one or more proteins of interest or therapeutic proteins, from the engineered bacteria. In the Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g. , OmpT cleavage thereby releasing the payload molecule(s) into the extracellular milieu (e.g., gut or tumor).

[0997] In some embodiments, the genetically engineered bacteria of the invention comprise a type III or a type Ill-like secretion system (T3SS) from Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The traditional T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. In the Type III traditional secretion system, the basal body closely resembles the flagella, however, instead of a "tail'Vwhip, the traditional T3SS has a syringe to inject the passenger proteins into host cells. The secretion tag is encoded by an N-terminal peptide (lengths vary and there are several different tags, see PCT/US 14/020972). The N-terminal tag is not removed from the polypeptides in this secretion system.

[0998] The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen, tumor microenvironment, or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest from the bacterial cytoplasm. In some embodiments, the secreted molecule, comprises a type III secretion sequence that allows the molecule of interest to be secreted from the bacteria.

[0999] In the Flagellar modified Type III Secretion, the tag is encoded in 5 'untranslated region of the mRNA and thus there is no peptide tag to cleave/remove. This modified system does not contain the "syringe" portion and instead uses the basal body of the flagella structure as the pore to translocate across both membranes and out through the forming flagella. If the fliC/fliD genes (encoding the flagella "tail'Vwhip) are disrupted the flagella cannot fully form and this promotes overall secretion. In some embodiments, the tail portion can be removed entirely.

[01000] In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[01001] For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173-bp region, is fused to the gene encoding the heterologous protein or peptide can be used to secrete polypeptides of interest (See, e.g., Majander et al., Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol. 2005 Apr;23(4):475-81). In some cases, the untranslated region from the fliC loci may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example, by using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g. , Duan et al., Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 23 7437-7438).

[01002] In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane- spanning secretion system. Single membrane- spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP- binding cassette translocases, flagellum/virulence-related translocases, conjugation- related translocases, the general secretory system (e.g. , the SecYEG complex in E. coli), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g. , Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.

[01003] In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g. , therapeutic polypeptides) - particularly those of eukaryotic origin - contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

[01004] One way to secrete properly folded proteins in gram-negative bacteria- particularly those requiring disulphide bonds - is to target the reducing - environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These "leaky" gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a "leaky" or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at -500,000 copies per cell and functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. 1. Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover.

Removal of periplasmic proteases such as degS, degP or nlpl can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes, in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some

embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl genes.

[01005] To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl, under the control of an inducible promoter. For example, expression of lpp or other cell wall stability protein or periplasmic protease can be repressed in conditions where therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., overexpression of colicins or the third topological domain of TolA, wherein peptide overexpression can be induced in conditions in which therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

[01006] Table 35 and Table 36 below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

Table 35. Secretion systems for gram positive bacteria

Table 36. Secretion Systems for Gram negative bacteria

Protein secretary pathways (SP) in gram-negative bacteria and their

descendants Type Name TC#² Bact Arch Eukarya # Energ

(Abbreviat eria aea Protei y ion) ns/Sys Sourc tern e

IMPS - Gram-negative bacterial inner membrane channel-forming translocases

ABC ATP binding 3.A.1 + + + 3-4 ATP (SIP) cassette

translocase

SEC General 3.A.5 + + + - 12 GTP (IISP) secretory OR translocase ATP

+

PMF

Fla/Path Flagellum/vir 3.A.6 + >10 ATP (IIISP) ulence- related

translocase

Conj Conjugation- 3.A.7 + >10 ATP (IVSP) related

translocase

Tat (IISP) Twin- 2.A.6 + + + 2-4 PMF arginine 4 (chloroplas

targeting ts)

translocase

Oxal Cytochrome 2.A.9 + + + 1 None (YidC) oxidase (mitochon or biogenesis dria PMF family chloroplast

s)

MscL Large 1.A.2 + + + 1 None conductance 2

mechanosens

itive channel

family

Holins Holin 1.E.1 + 1 None functional •21

superfamily

Eukaryotic Organelles

MPT Mitochondria 3.A. + >20 ATP

1 protein B (mitochon

translocase drial)

CEPT Chloroplast 3.A.9 (+) + >3 GTP envelope (chloroplas

protein ts)

translocase Bcl-2 Eukaryotic 1.A.2 + 1? None Bcl-2 family 1

(programmed

cell death)

Gram-negative bacterial outer membrane channel-forming translocases

MTB Main 3.A.1 - 14 ATP; (IISP) terminal 5 PMF branch of the

general

secretory

translocase

FUP AT- 1 Fimbrial 1.B.1 +^b None usher protein 1 +^b - None

^!

Autotransport 1.B.1

er- 1 2

AT-2 Autotransport 1.B.4 +^b None OMF er-2 0 +^b +(?) None

^!

(ISP) 1.B.1

7

TPS 1.B.2 + + None Secretin 0 +^b None

^!

(IISP and 1.B.2

IISP) 2

OmpIP Outer 1.B.3 + + >4 None membrane 3 (mitochon ? insertion dria;

porin chloroplast

s)

[01007] The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe / Volume 1, Number 9, 2006 "Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently", the contents of which is herein incorporated by reference in its entirety.

[01008] In some embodiments, the genetically engineered bacterial comprise a native or non-native secretion system described herein for the secretion of anti-cancer molecule (e.g. , a kynureninase or others described herein), a gut barrier enhancer molecule, an ant i- inflammatory molecule, or a satiety effector or a different molecule described herein. Table 37A. Polypeptide Sequences of exemplary secretion tags

Table 31C. Additionals secretion tag sequences (native to E coli.)

[01009]

[01010] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that encodes a polypeptide which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 1511, SEQ ID NO: 1512, SEQ ID NO: 1513, and/or SEQ ID NO: 1514.

[01011] Any secretion tag or secretion system can be combined with any cytokine described herein, and can be used to generate a construct (plasmid based or integrated) which is driven by a directly or indirectly inducible or constitutive promoter described herein. In some embodiments, the secretion system is used in combination with one or more genomic mutations, which leads to the leaky or diffusible outer membrane phenotype (DOM), including but not limited to, lpp, nlP, tolA, PAL.

[01012] In some embodiments, the secretion system is selected from the type III flagellar, modified type III flagellar, type I (e.g. , hemolysin system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, a single membrane secretion system, Sec and, TAT secretion systems.

[01013] Any of the secretion systems described herein may according to the disclosure be employed to secrete the polypeptides of interest. In some

embodiments, therapeutic proteins secreted by the genetically engineered bacteria are modified to increase resistance to proteases, e.g. intestinal proteases.

Table 38. Comparison of Secretion systems for secretion of polypeptide from engineered bacteria

[01014] In some embodiments, therapeutic polypeptides of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a therapeutic polypeptide of interest, such as, kynureninase, or any other tryptophan synthesis or catabolism enzymes described herein, is assembled behind a fliC-5'UTR (e.g. , 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the expression of therapeutic peptide of interested secreted using components of the flagellar type III secretion system is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g. , FNR- inducible promoter), promoters induced by tumor specific specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g. , arabinose is used. In some embodiments, therapeutic polypeptide of interest is expressed from a plasmid (e.g. , a medium copy plasmid). In some embodiments, therapeutic polypeptide of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g. , the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency.

[01015] In some embodiments, therapeutic polypeptides of interest, e.g. , kynureninase, or any other tryptophan synthesis or catabolism enzymes described herein, are secreted using via a diffusible outer membrane (DOM) system. In some embodiments, therapeutic polypeptide of interest is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, therapeutic polypeptide of interest is fused to a Tat-dependent secretion signal. Exemplary Tat- dependent tags include TorA, FdnG, and DmsA.

[01016] In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include lpp, pal, tolA, and/or nlpl. In some embodiments, lpp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, tolA is deleted or mutated. In other embodiments, nlpl is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g., to increase stability of the polypeptide in the periplasm. Non- limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated.

[01017] In some embodiments, therapeutic polypeptides of interest, e.g., kynureninase, or any other tryptophan synthesis or catabolism enzymes described herein, are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodiments, therapeutic protein of interest is expressed as a fusion protein with the native Nissle auto-secreter E. co/z_01635 (where the original passenger protein is replaced with therapeutic polypeptides of interest.

[01018] In some embodiments, therapeutic polypeptides of interest, e.g. kynureninase, or any other tryptophan synthesis or catabolism enzymes described herein, are secreted via Type I Hemolysin Secretion. In some embodiments, therapeutic polypeptide of interest is expressed as fusion protein with the 53 amino acids of the C terminus of alpha-hemolysin (hlyA) of E. coli CFT073.

[01019] In some embodiments, the gene sequences encoding the polypeptide of interest for secretion are operably linked to one or more directly or indirectly inducible promoter(s). In some embodiments, the gene sequences encoding the polypeptide of interest for secretion are operably linked to a directly or indirectly inducible promoter that is induced under exogeneous environmental conditions, e.g., conditions found in the gut, the tumor microenvironment or other tissue specific conditions. In some embodiments, the gene sequences encoding the polypeptide of interest for secretion are operably linked to a directly or indirectly inducible promoter that is induced by metabolites found in the gut, the tumor microenvironment or other specific conditions. In some embodiments, the gene sequences encoding the polypeptide of interest for secretion are operably linked to a directly or indirectly inducible promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene sequences encoding the polypeptide of interest for secretion are operably linked to a directly or indirectly inducible promoter that is induced under inflammatory conditions (e.g., RNS, ROS), as described herein. In some embodiments, the gene sequences encoding the polypeptide of interest for secretion are operably linked to a directly or indirectly inducible promoter that is induced under immunosuppressive conditions, e.g., as found in the tumor, as described herein. In some embodiments, two or more gene gene(s) are linked to a directly or indirectly inducible promoter that is induced by exposure a chemical or nutritional inducer, which may or may not be present under in vivo conditions and which may be present during in vitro conditions (such as strain culture, expansion, manufacture), such as tetracycline or arabinose, or others described herein. In some embodiments, the two or more payloads are all linked to a constitutive promoter. Such constitutive promoters are described in Table 10 - Table 20 herein. In some embodiments, the two or more gene sequence are operably linked to the same promoter sequences. In some embodiments, the two or more gene sequence are operably linked to two or more different promoter sequences, which can either all be constitutive (same or different constitutive promoters), all inducible (by same or different inducers), or a mix of constitutive and inducible promoters.

[01020] In some embodiments, the one or more nucleic acid sequence(s) encoding one or more polypeptides of interest for secretion is located on a plasmid in the bacterial cell. In another embodiment, the one or more nucleic acid sequence(s) encoding one or more payload molecules is located in the chromosome of the bacterial cell.

[01021] In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganisms chromosome. Also, in some embodiments, the genetically engineered microorganisms are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g. , thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art and (7) one or more circuits for the production or degradation of one or more metabolites (e.g., kynurenine, tryptophan, adenosine, arginine) described herein (8) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art and (9) combinations of one or more of such additional circuits.

Surface Display

[01022] In some embodiments, the genetically engineered bacteria and/or microorganisms encode one or more gene(s) and/or gene cassette(s) encoding a polypeptide of interest described herein which is anchored or displayed on the surface of the bacteria and/or microorganisms. Examples of the payload molecules which are displayed or anchored to the bacteria and/or microorganism, are any of the payload molecules or other effectors described herein, and include but are not limited to enzymes (e.g., kynureninase), antibodies, e.g. , scFv fragments, and tumor- specific antigens or neoantigens. In a non-limiting example, the payloadwhich are anchored or displayed on the bacterial cell surface are directed against checkpoint inhibitors described herein, including, but not limited to, CLTLA4, PD- 1, PD-L1, and others described herein.

[01023] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding therapeutic polypeptide or effector molecule, e.g. , a kynureninase or a scFv, which is anchored or displayed on the surface of the bacteria, and which remains anchored while exerting its effector function. In other embodiments, the genetically engineered bacteria encoding the surface-displayed therapeutic polypeptide, e.g., the a kynureninase or antibodies or scFv fragments or other effectors described herein, lyse before, during or after exerting their effector function. In some embodiments, the genetically engineered bacteria encode a therapeutic peptide that is temporarily attached to the cell surface and which dissociates from the bacterium before, during, or after exerting its function. [01024] In some embodiments, shorter peptides or polypeptides, e.g. peptides or polypeptides of less than 60 amino acids of length, are displayed on the cell surface of the genetically engineered bacteria. In some embodiments, such shorter peptides or polypeptides comprise a immune modulatory effector molecule. Non- limiting examples of such therapeutic polypeptides are described herein.

[01025] Several strategies for the display of shorter peptides or polypeptides on the surface of gram negative bacteria are known in the art, and are for example described in Georgiou et al., Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines: Nat Biotechnol. 1997 Jan;15(l):29-34, the contents of which is herein incorporated by reference in its entirety. These systems all share a common theme, targeting recombinant proteins to the cell surface by the construction of gene fusions using sequences from membrane- anchoring domains of surface proteins.

[01026] Non-limiting examples of such strategies are described in Table

63A and Table 63B, and Table 63C.

Table 39A. Exemplary Cell Surface Display Strategies

Flagellin E. coli Sandwich Cell surface (FliC) fusion

Flagellin E. coli Sandwich Cell surface (FliC) fusion

FimH (type I E. coli Sandwich Cell surface pili) fusion

PapA (Pap E. coli Sandwich Cell surface pili) fusion

PulA Klebsiella C-terminal Cell

fusion surface/extracellular fluid

Table 39B. Exemplary Cell Surface Display Strategies

Subunits of Surface

Appendages

Flagellae 11-115 aa

Fimbriae 7-52 aa

S-layer proteins

RsaA 12 aa

Table 39C. Exemplary Cell Surface Strategies

Invasin C-terminal 1.1

MSPla N-terminal 4.6

[01027] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more short therapeutic peptides or polypeptides fused into surface exposed loops of outer membrane proteins (OMPs), e.g., from enteric bacteria. In a non- limiting example, the short therapeutic peptides or polypeptides expressed by the genetically engineered bacteria are inserted into the outer membrane protein LamB, e.g., from E. coli, and displayed on the bacterial cell surface. Extracellular display of peptides through insertion of peptides into surface exposed loops of LamB is for example described in Hofnung et al., Expression of foreign polypeptides at the Escherichia coli cell surface; Methods Cell Biol. 34:77-105, and Charbit, A. et al., 1987. Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria, J. Immunol. 139: 1658-1664.

[01028] In another non-limiting example, the short therapeutic peptides or polypeptides encoded by one or more gene sequence(s) comprised in the genetically engineered bacteria are inserted into the outer membrane protein PhoE, e.g., from E. coli, and displayed on the bacterial cell surface. The PhoE protein is another abundant outer membrane protein of E. coli K-12, which has a trimeric structure and functions as a pore for small molecules. Analysis of the primary structure of PhoE revealed 16 beta sheets which traverse through the membranes, and eight hypervariable regions exposed at the surface of the cell. One or more of these cell surface exposed regions of PhoE protein can be used to insert heterologous peptides. For example, antigenic determinants of pathogenic organisms have been presented in one or more cell surface exposed regions of PhoE protein {e.g., as described in Aterberg et al., 1990; Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants:

immunogenicity of epitopes of foot-and-mouth disease virus; Vaccine. 1990

Feb;8(l):85-91).

[01029] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more short therapeutic peptides or polypeptides fused to protein components of extracellular appendages. Several systems have been described, in which extracellular appendages, such as pili and flagella are used to display peptides of interest at the bacterial cell surface. Examples of flagellar and pilar proteins used include FliC, a major structural component of the E. coli flagellum, and PapA, the major subunit of the Pap pilus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more components of a FLITRX system. The FLITRX system is an E. coli display system based on the use of fusion protein of FliC and thioredoxin, a small redox protein which represents a highly versatile scaffold that allows peptide inserts to assume a confirmation compatible with binding to other proteins. In the FLITRX system, thioredoxin is fused into a dispensable region of FliC. Then, heterologous peptides can be inserted within the thioredoxin domain in the FliC fusion, and are surface exposed. Other scaffolding proteins are known in the art, some of which may replace thioredoxin as a scaffolding protein in this system.

[01030] In some embodiments, the genetically engineered bacteria comprise a FimH fusion protein, in which therapeutic peptide of interest is fused to FimH, an adhesin of type 1 fimbriae, e.g., from E. coli. FimH adhesin chimeras containing as many as 56 foreign amino acids in certain positions are transported to the bacterial surface as components of the fimbrial organelles (Pallesen et al., Chimeric FimH adhesion of type I fimbriae: a bacterial surface display system for heterologous sequences. Microbiology 141: 2839-2848).

[01031] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a fusion protein in which therapeutic peptide of interest is fused to the major subunit of Fl l fimbriae, e.g., from E. coli. Hypervariable regions of the major subunit of Fl 1 fimbriae can be used for insertion of heterologous peptides, e.g., antigenic epitopes (Van Die et al., Expression of foreign epitopes in P-fimbriae of Escherichia coli. Mol. Gen. Genet. 222: 297-303).

[01032] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a papA fusion protein, in which therapeutic peptide of interest is fused to papA. In some embodiments, peptides of interest are inserted following either codon 7 or 68 of the coding sequence for the mature portion of PapA, as peptides in the area of amino acids 7 and 68 of PapA are localized at the external side of the pilus (Steidler et al., Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of

Staphylococcus aureus in Escherichia coli; J Bacteriol. 1993 Dec;175(23):7639-43). [01033] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s), which encode polypeptides larger than 60 amino acids, e.g., immune modulatory effector, and which are displayed on the bacterial cell surface. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s), which encode a fusion protein, in which a therapeutic peptide of interest, e.g., a polypeptide greater than 60 amino acids in length, is fused to a lipoprotein from a gram negative bacterium, or one or more fragments thereof.

[01034] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s), which encode a fusion protein, in which a therapeutic protein of interest is fused to peptidoglycan associated lipoprotein (PAL) or a fragment thereof. The fusion protein in located in the periplasm and can be displayed externaly upon permeablization of the outer membrane. For example, a PAL-scFv fusion protein was shown to bind its antigen and to be tightly bound to the murein layer of the cell envelope (Fuchs et al., Targeting recombinant antibodies to the surface of Escherichia coli fusion to a peptidoglycan-associated lipoprotein; Biotechnology (N Y). 1991 Dec;9(12): 1369-72). The PAL-scFv fusion was located in the periplasm and bound to the murein layer, and after permeabilization of the outer membrane, the scFv became accessible to externally added antigen. In some embodiments, the genetically engineered bacteria comprising a fusion protein for surface display further have a permeable outer membrane. Mutations and/or deletions resulting in a leaky outer membrane are described elsewhere herein.

[01035] In some embodiments, the genetically engineered bacteria encode a fusion protein, in which a therapeutic protein of interest, e.g., a immune modulatory effector, is fused to residues of the major lipoprotein of a gram negative bacterium, e.g., E. coli. In some embodiments, the genetically engineered bacteria encode a fusion protein, in which a therapeutic protein of interest, is fused to the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of a gram negative bacterium, e.g., E. coli. These residues of the E. coli major lipoprotein function as a hydrophobic membrane anchor. For example, a fusion construct of these residues with a therapeutic polypeptide, in this case a scFv fragment, resulted in specific accumulation of an immunoreactive and cell- bound polypeptide in E. coli (Laukkanen et al., Lipid- tagged antibodies: bacterial expression and characterization of a lipoprotein- single- chain antibody fusion protein. Mol. Microbiol. 4: 1259-1268). [01036] In some embodiments, the genetically engineered bacteria encode a fusion protein, in which a therapeutic protein of interest, is inserted into the TraT protein of a gram negative bacterium, e.g., E. coli, e.g. at position 180. The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. Taylor et al. showed that insertion of the C3 epitope of polio virus, e.g., at position 180, allowed exposure of the antigen to the cell surface, while the oligomeric conformation of the wild-type protein was maintained (Taylor et al., The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure-function relationship in the TraT protein. Mol Microbiol. 1990 Aug;4(8): 1259-68).

[01037] In some embodiments, the genetically engineered bacteria comprise one or more genes and/or gene cassettes encoding a fusion protein comprising a Lpp-OmpA display vehicle comprising the N terminal outer membrane signal from the major lipoprotein (Lpp) fused to a domain from the outer membrane protein OmpA, fused to therapeutic polypeptide of interest. In this system, the Lpp signal peptide mediates localization, and OmpA provides the framework for the display of therapeutic protein of interest. Lpp-OmpA fusions have been used to display several proteins between 20 and 54 kDa in size on the surface of E. coli (see, e.g., Staphopoulos et al., Characterization of Escherichia coli expressing and Lpp-OpmA (46-159)-PhoA fusion protein localized in the outer membrane). For example, Fransco et al fused beta - lactamase to the N-terminal targeting sequence of Lpp and an OmpA fragment containing 5 of the 8 membrane spanning loops of the native protein. This fusion protein was assembled on the cell surface and the beta- lactamase domain was stably anchored in the cell wall (Fransisco et al., Transport ansd anchoring of beta- lactamase to the external surface of Escherichia coli; Proc. Natl. Acad. Sci. USA Vol 89, pp. 2713- 2717, 1992).

[01038] In some embodiments, the Type II secretion pathway or a variation thereof is used to for transient or longer duration display of therapeutic proteins of interest on the bacterial cell surface, e.g., the IgA protease secretion pathway of Neisseria or the VirG protein pathway of Shigella. In some embodiments, the IgA protease secretion pathway is used to export and display therapeutic peptides of interest on the cell surface of gram negative bacteria. The IgA proteases of Neisseria

gonorrhoeae and Hemophilus influenza use a variation of the most common, Type II secretion pathway, to achieve extracellular export independent of any other gene products. The IgA genes of Neisseria species encode extracellular proteins that cleave human IgAl antibody. The iga gene alone is sufficient to direct selected extracellular secretion of IgA protease in Neisseria, Salmonella, and E. coli species (Klauser et al., 1993, Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. EMBO J 9: 1991- 1999, and references therein). The mature IgA protease is processed in several steps from a large precursor by signal peptidase and autoproteolytic cleavage. The precursor consists of four domains: (1) an amino terminal signal peptide which mediates inner membrane transport; (2) the protease domain (3) the alpha domain, a basic alpha helical region which is secreted with the protease and (4) the autotransporter beta domain which harbors the essential function for outer membrane transport. Essentially, the C- terminal beta autotransporter domain of the IgA protease forms a channel in the outer membrane that mediates the export of the N terminal domain across the membrane, which in turn becomes transiently displayed on the external surface of the bacteria. The alpha domain and protease domain are then released through proteolytic cleavage. Klauser et al. (1993), showed that replacement of the native N-terminal domains of IgA protease of N. gonorrhoeae with the cholera toxin B resulted in the surface presentation of the passenger polypeptide in S. typhymurium. In another study, the signal sequence and the C-terminal beta autotransporter domain of the IgA protease of Neisseria gonorrhoeae was used to translocate and display a scFv directed against a porcine epidemic diarrhea virus epitope on the bacterial cell surface of E. coli (Pyo et al., Escherichia coli expressing single chain Fv on the cell surface as a potential

prophylactic of porcine epidemic diarrhea virus; Vaccine (27) (2009) 2030-2036.).

[01039] Thus, In some embodiments, the genetically engineered bacteria encode a IgA protease fragment in which the alpha domain is substituted with a therapeutic protein of interest, and fused to a functional IgA protease beta-domain, which mediates export through the outer membrane. Without wishing to be bound by theory, IgA protease activity is eliminated in such a fusion protein, and therefore the autoproteoulytic release of the fusion protein into the medium does not occur, resulting in the display of therapeutic protein of interest on the cell surface of the gram-negative host bacterium. [01040] The secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria (see., e.g. , Suzuki et al., 1995; Extracellular transport of VirG protein in Shigella J Biol. Chem 270:30874-30880, and references therein). Thus, in some embodiments, the genetically engineered bacteria encode a fusion protein comprising a therapeutic protein of interest fused to the membrane spanning region of VirG, resulting in surface display of therapeutic protein of interest. The VirG gene on the large plasmid of Shigella has been shown to be responsible for the localized deposition of filamentous actin (F-actin) trailing from one pole of invading bacterial cells and extending in a filament through the host epithelial cytoplasm. VirG is a surface-exposed outer membrane protein consisting of three distinctive domains, the N-terminal signal sequence (amino acids 1-52), the id a-domain (amino acids 53-758), and the dC-terminal β-core (amino acids 759- 1102) (see, e.g. , Suzuki et al., 1996; Functional Analysis of Shigella VirG Domains Essential for Interaction with Vinculin and Actin-based Motility; J. Biol. Chem., 271, 21878-21885, and references therein). Suzuki et al. (1995); showed that the fusion of a foreign protein such as MalE or PhoA protein to the N terminus 37-kDa VirG portion resulted in the transport of the passenger polypeptides from the periplasm to the external side of the outer membrane, indicating that the C-terminal 37-kDa VirG portion embedded in the outer membrane is involved in the translocation of the preceding VirG portion or the heterologous or passenger polypeptide from the periplasmic space to the external side of the outer membrane, in a manner homologous to the IgA protease beta-domain. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a fusion protein, in which a C-terminal 37-kDa VirG protein fragment is fused to a therapeutic protein of interest.

[01041] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a fusion protein, in which a therapeutic protein of interest is fused to pullulanase for temporary surface display. Pullulanase is specifically released into the medium by Klebsiella pneumonieae, and exists as a fully exposed, cell surface-bound intermediate before it is released into the medium from early stationary growth phase onwards. Cell-surface anchoring is accomplished by an N-terminal fatty acyl modification whose chemical composition is identical to that of other bacterial protein. [01042] Unlike the IgA protease, the lipoprotein pullulanase (PulA) of

Klebsiella pneumoniae, which is also exported via a type II secretion mechanism, requires 14 genes for its translocation across the outer membrane. For example, Pugsley and coworkers have shown that the lipoprotein pullulanase (PulA) can facilitate translocation of the periplasmic enzyme beta-lactamase across the outer membrane. In particular, in E. coli strains expressing all pullulanase secretion genes, pullulanase-beta- lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently transported to the cell surface. Of note, pullulanase hybrids remain only temporarily attached to the bacterial surface and are subsequently released into the medium (Kornacker and Pugsley: The normally periplasmic enzyme beta-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell surface enzyme pullulanase. Mol.

Microbiol. 4: 1101-1109, and references therein). Accordingly, in some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising a complete set of pullulanase genes required for secretion and fusion protein comprising a therapeutic protein of interest fused to a N-terminal pullulanase polypeptide fragment, e.g., as described by Kornacker and Pugsley. In some embodiments, the fusion proteins comprising N-terminal pullulanase polypeptide fused to therapeutic protein of interest, are transiently displayed on the surface of the bacterial cell, and subsequently released into the media or extracellular space.

[01043] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a fusion protein in which the ice nucleation protein (INP) from Pseudomonas syringae anchors a therapeutic protein of interest in the cell wall. INP is a secretory protein that catalyzes extracellular ice formation as the ice nuclei. INP has been found in a number of Gram-negative species, including P. syringae, Erwinia herbicola, Xanthomonas campestris, and Pseudomonas fluorescens. Four genes in P. syringae strains , inaK, inaV , and inaZ , and inaQ exhibit high similarities in sequences and in primary organization (Li et al., Molecular

Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli Int J Biol Sci. 2012; 8(8): 1097-1108). All INPs (1200 aa to 1500 aa) comprise of three distinct structural domains: (1) the N-terminal domain (approximately 15% of the total sequence), which is relatively hydrophobic and which is are potentially capable of being coupled to the mannan-phosphatidylinositol group in the outer membrane through N- glycan (Asp) or Oglycan (Ser, Thr) linkages; (2) the C-terminal domain (approximately 4%), which is a relatively hydrophilic terminus; and (3) the central repeating domain (CRD) (approximately 81%), which constitutes contiguous repeats given by 16-residue (or 48-residue) periodicities with a consensus octapeptide (Ala-Gly-Tyr-Gly-Ser-Thr- Leu-Thr). INPs have been employed in various bacterial cell- surface display systems including E. coli, Zymomonas mobilis, Salmonella^ sp., Vibrio anguillarum,

Pseudomonas putida, and cyanobacteria, in all od which INPs were able to target a heterologous protein onto the surface of the host cell. Moreover, the N-terminal region alone was shown to direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif (Li et al., 2004 Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein; Biotechnol Bioeng. 2004 Jan 20;85(2):214-21). Accordingly, in some embodiments, the genetically engineered bacteria comprise IMP fusions for surface display of a therapeutic peptide of interest. In some embodiments, the N-terminal region of the INP protein is fused to the polypeptide of interest for surface display.

[01044] IMP proteins further have modifiable internal repeating units, ie.,

CRD length is adjustable, which is allows flexibility in protein fusion length (Jung et al., 1998), and also can accommodate larger polypeptides. For example, the INP-based display systems were used to successfully express a 90 kDA protein on the cell surface of E. coli (Wu et al., 2006; Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity; FEMS Bicrobiology Letters, Volume 256, Issue 1; Pages 119-125).

[01045] It is understood by those skilled in the art that translocation of such fusion or hybrid proteins described herein requires a "translocation-competent" conformation, e.g., the formation of disulfide bonds, e.g., in the periplasmic space, may be undesirable and inhibit translocation through the outer membrane (see, e.g., Klauser et al., 1990), or alternatively may be required for, (or at least not impede) translocation through the outer membrane (see, e.g., Puggsley, 1992; Translocation of a folded protein across the outer membrane in Escherichia coli; Proc Natl Acad Sci U S A. 1992 Dec 15; 89(24): 12058-12062). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding for a fusion protein in which disulfide bonds are prevented from forming prior to the translocation to the cell surface. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding for a fusion protein in which disulfide bonds are formed prior to translocation to the cell surface.

[01046] Expression systems for the display of proteins in Gram-positive bacteria have also been developed. Consequently, in some embodiments, gram positive bacteria are engineered to display therapeutic proteins of interest on their cell surface. Uhlen et al. used fusions to the cell-wall bound, X-domain of protein A, for the display of foreign peptides up to 88 amino acids long to the surface of Staphylococcus strains. For example one study describes an expression system to allow targeting of

heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase- negative gram-positive bacterium (Hansson et al., Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus; J Bacteriol. 1992 Jul;174(13):4239-45).

[01047] The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane- anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. Accordingly, in some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a therapeutic polypeptide fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A

[01048] E. co/z-staphylococcus shuttle vectors have been constructed by taking advantage of the promoter, signal sequence, and propeptide region from the lipase gene construct derived from S. hyicus and the cell surface attachment part of staphylococcal protein A. This system has been investigated for the surface display of heterologous polypeptides on S. carnosus (Samuelson et al., Cell surface display of recombinant proteins on Staphylococcus carnosus; J Bacteriol. 1995 Mar;177(6): 1470- 6). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a therapeutic polypeptide fusion protein comprising promoter, signal sequence, and propeptide region from the lipase gene construct derived from S. hyicus and the cell surface attachment part of staphylococcal protein A.

[01049] In other studies, the fibrillary M6 proteins of Streptococcus pyrogenes was employed as a carrier for antigen delivery in Streptococcus cells. (Pozzi et al., 1992; Delivery and expression of a heterologous antigen on the surface of streptococci. Infect. Immunm. 60: 1902-1907). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising therapeutic polypeptide fusion proteins comprising the fibrillary M6 proteins of Streptococcus pyrogenes for cell surface display of therapeutic polypeptide.

[01050] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with an intimin or invasin.

Intimins and invasins belong to a family of bacterial adhesins which specifically interact with various eukaryotic cell surface receptors, thereby mediating bacterial adherence and invasion. Both intimins and invasins provide a structural scaffold ideally suited to the cell surface display.

[01051] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with an intimin, e.g., with the Enterohemorragic E. coli Intimin EaeA protein or a carboxy-terminal truncation thereof {e.g., as described inWentzel et al, Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA J Bacteriol. 2001 Dec; 183(24): 7273-7284). For example, N-terminal 489 amino acids of invasin are sufficient to promote the localization of a fusion protein to the cell surface. [030] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with an invasin, e.g. Enterohemorrhagic E. coli invasion, or a carboxyterminal truncation thereof. For example, N-terminal 539 amino acids of intimin were sufficient to promote outer membrane localization of a fusion protein ( Liu et al., The Tir-binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial-induced host cell signaling; Mol Microbiol. 1999 Oct;34(l):67-81).

[01052] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with Bacillus anthracis exosporal protein (BclA) as an anchoring motif. The BclA is an exosporium protein, a hair- like protein surrounding the B. anthracis spore. In a nonlimiting example, a polypeptide of interest is linked to the C-terminus of N-terminal domain (21 amino acids) of BclA, e.g. , as described in Park et al. (Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis).

[01053] Various other anchoring motifs have been developed including

OprF. QmpC, and QmpX. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with OprF, OmpC, and OmpX.

[01054] In some embodiments, therapeutic polypeptides of interest are permanently displayed on the cell surface of the genetically engineered bacterium. In some embodiments, therapeutic polypeptides of interest are transiently displayed on the cell surface of the genetically engineered bacterium.

[01055] In some embodiments, therapeutic polypeptides are displayed in strains, e.g., described herein which display a leaky phenotype. Such strains have deactivating mutations in one or more of genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl.

[01056] In some embodiments, one or more ScFvs are displayed on the bacterial cell surface, alone or in combination with other therapeutic polypeptides of interest. In some embodiments, one or more tryptophyan catabolism enzymes, e.g., kynureninase, are displayed on the bacterial cell surface, alone or in combination with other therapeutic polypeptides of interest.

[01057] In some embodiments, a cell surface display strategy or circuit is combined with a secretion strategy or circuit in one bacterium. In some embodiments, the same polypeptide is both displayed and secreted. In some embodiments, a first polypeptide is displayed and a second is secreted. In some embodiments, a display strategy or circuit strategy is combined with a circuit for the intracellular production of an enzyme and consequentially intracellular catabolism of its substrate. In some embodiments, a display strategy or display circuit is combined with a circuit for the intracellular production of a gut barrier enhancer molecule and/or an ant i- inflammatory effector molecule.

[01058] In some embodiments, the expression of the surface displayed polypeptide or fusion protein is driven by an inducible promoter. In some

embodiments, the inducible promoter is an oxygen level-dependent promoter (e.g., FNR- inducible promoter). In some embodiments, the inducible promoter is induced by gut- specific and/or tumor- specific or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), or promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g. , arabinose. In alternate embodiments, expression of the surface displayed polypeptides or polypeptide fusion proteins is driven by a constitutive promoter.

[01059] In some embodiments, the expression of the surface displayed polypeptide or fusion protein is plasmid based. In some embodiments, the gene sequence(s) encoding the payload for surface display is chromosomally inserted. Table 40. Lists Selected display anchors

Table 40. Selected display anchors

[01060] In some embodiments, the display anchor is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%

homologous to the sequence of SEQ ID NO: 48, SEQ ID NO: 49, and/or SEQ ID NO: 50. [01061] Any of the polypeptides of interest or payloads described herein can be displayed on the cell surface of the genetically engineered bacteria using any of the display systems and anchors described herein. In some embodiments, the gene sequences encoding the polypeptide of interest, e.g., kynureninase, a anti-PD-1 antibody, or any other tryptophan synthesis or catabolism enzymes described herein, for anchored display are operably linked to one or more directly or indirectly inducible promoter(s). In some embodiments, the gene sequences encoding the polypeptide of interest for anchored display are operably linked to a directly or indirectly inducible promoter that is induced under exogeneous environmental conditions, e.g., conditions found in the gut, the tumor microenvironment or other tissue specific conditions. In some embodiments, the gene sequences encoding the polypeptide of interest for anchored display are operably linked to a directly or indirectly inducible promoter that is induced by metabolites found in the gut, the tumor microenvironment or other specific conditions. In some embodiments, the gene sequences encoding the polypeptide of interest for anchored display are operably linked to a directly or indirectly inducible promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene sequences encoding the polypeptide of interest for anchored display are operably linked to a directly or indirectly inducible promoter that is induced under inflammatory conditions (e.g., RNS, ROS), as described herein. In some embodiments, the gene sequences encoding the polypeptide of interest for anchored display are operably linked to a directly or indirectly inducible promoter that is induced under immunosuppressive conditions, e.g., as found in the tumor, as described herein. In some embodiments, two or more gene gene(s) are linked to a directly or indirectly inducible promoter that is induced by exposure a chemical or nutritional inducer, which may or may not be present under in vivo conditions and which may be present during in vitro conditions (such as strain culture, expansion, manufacture), such as tetracycline or arabinose, or others described herein. In some embodiments, the two or more payloads are all linked to a constitutive promoter. Such constitutive promoters are described in Table 10 - Table 20 herein. In some embodiments, the two or more gene sequence are operably linked to the same promoter sequences. In some embodiments, the two or more gene sequence are operably linked to two or more different promoter sequences, which can either all be constitutive (same or different constitutive promoters), all inducible (by same or different inducers), or a mix of constitutive and inducible promoters.

[01062] In some embodiments, the one or more nucleic acid sequence(s) encoding one or more polypeptides of interest for anchored display is located on a plasmid in the bacterial cell. In another embodiment, the one or more nucleic acid sequence(s) encoding one or more payload molecules is located in the chromosome of the bacterial cell.

[01063] In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganisms chromosome. Also, in some embodiments, the genetically engineered microorganisms are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art and (7) one or more circuits for the production or degradation of one or more metabolites (e.g., kynurenine, tryptophan, adenosine, arginine) described herein (8) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art and (9) combinations of one or more of such additional circuits.

Essential Genes and Auxotrophs

[01064] As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et ah, Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).

[01065] An "essential gene" may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the recombinant bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.

[01066] Exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain are shown below. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.

[01067] n auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some

embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In some embodiments, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil, as long as the corresponding wild-type gene product is not produced in the bacteria. Table 9 lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis. Table 9. Non-limiting Examples of Bacterial Genes Useful for Generation of an Auxotroph

glnA uraA dapB

ilvD dapD

leuB dapE

lysA dapF

serA

metA

glyA

hisB

ilvA

pheA

pro A

thrC

trpC

tyrA

[01068] Table 10 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.

Table 10. Survival of amino acid auxotrophs in the mouse gut

hisB Histidine Present Present Present ilvA Isoleucine Present Present Absent leuB Leucine Present Present Absent lysA Lysine Present Present Absent metA Methionine Present Present Present pheA Phenylalanine Present Present Present pro A Proline Present Present Absent serA Serine Present Present Present thrC Threonine Present Present Present trpC Tryptophan Present Present Present tyrA Tyrosine Present Present Present ilvD Valine/lsoleucine/ Present Present Absent

Leucine thyA Thiamine Present Absent Absent uraA Uracil Present Absent Absent flhD FlhD Present Present Present

[01069] For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[01070] Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine bio synthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[01071] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[01072] In complex communities, it is possible for bacteria to share DNA.

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

[01073] Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, UgA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, Igt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, IpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, IpxA, IpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, Int, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, IpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabl, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

[01074] In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson "Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain,"ACS Synthetic Biology (2015) DOI:

10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

[01075] In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG, and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS

comprising one or more of the following mutations: L36V, C38A, and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A, and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I, and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I, and L6G.

[01076] In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole- 3 -acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnciN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2- aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I, and L6G) are complemented by benzothiazole or indole.

[01077] In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).

[01078] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system.

[01079] In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the

recombinant bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein and in Figs. 29-32. Other embodiments, are described in Wright et al., "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synthetic Biology (2015) 4: 307-316, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill- switch circuitry, such as any of the kill- switch components and systems described herein, as well as another bio security system, such a conditional origin of replication (Wright et al., 2015).

[01080] In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that consume excess ammonia. In a more specific aspect, auxotrophic modifications may be used to screen for mutant bacteria that consume excess ammonia by overproducing arginine. As described herein, many genes involved in arginine metabolism are subject to repression by arginine via its interaction with ArgPv. The astC gene promoter is unique in that the arginine- ArgR complex acts as a transcriptional activator, as opposed to a transcriptional repressor. AstC encodes succinylornithine aminotransferase, the third enzyme of the ammonia-producing arginine succinyltransferase (AST) pathway and the first of the astCADBE operon in E. coli (Schneider et al., 1998). In certain embodiments, the genetically engineered bacteria are auxotrophic for a gene, and express the auxotrophic gene product under the control of an astC promoter. In these embodiments, the auxotrophy is subject to a positive feedback mechanism and used to select for mutant bacteria which consume excess ammonia by overproducing arginine.

Genetic Regulatory Circuits

[01081] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g. , kynureninease, and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the payload is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the payload is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

[01082] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g. , kynureninease, and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the tetR, and the payload is expressed.

[01083] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g. , kynureninease, and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.

[01084] Examples of repressors useful in these embodiments, include, but are not limited to, ArgR, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

[01085] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g. , kynureninease, and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a payload. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the payload. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing payload translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.

[01086] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g. , kynureninease, and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed.

[01087] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g. , kynureninease, and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a payload operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the payload remains in the 3' to 5' orientation, and no functional payload is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the payload is reverted to the 5' to 3' orientation, and functional payload is produced. [01088] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload, e.g., kynureninease, and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload. The third gene encoding the T7 polymerase is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the

recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR- responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3' to 5' orientation, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5' to 3' orientation, and the payload is expressed.

Host-Plasmid Mutual Dependency

[01089] In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al, 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad- spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention.

[01090] The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

[01091] Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a gut enhancer molecule and further comprise a toxin-anti-toxin system that simultaneously produces a toxin (hok) and a short-lived anti-toxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015). In some embodiments, the genetically engineered bacteria further comprise alp7 from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015).

Kill Switch

[01092] In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos.

62/183,935, 62/263,329, and 62/277,654, each of which is incorporated herein by reference in their entireties). The kill switch is intended to actively kill engineered microbes in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

[01093] Bacteria engineered with kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a bio fuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease or disorder may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a therapeutic gene(s) or after the subject has experienced therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of arg^A^^r. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of arg^A^^r, for example, after the production of arginine or citrulline.

Alternatively, the bacteria may be engineered to die after the bacteria has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject). Examples of such toxins that can be used in kill- switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et ah, 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl β-D-l-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et ah, 2010). In some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of arg^A:fb . In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of argA^. [01094] Kill- switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g. , the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.

[01095] Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous

environmental signal, for example, in a low-oxygen environment. In some

embodiments, the genetically engineered bacteria of the present disclosure, e.g. , bacteria expressing argA^ and repressor ArgR, comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In some embodiments, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable.

[01096] In another embodiment in which the genetically engineered bacteria of the present disclosure, e.g. , bacteria expressing argA^ and repressor ArgR, express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In some embodiments, the at least one

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

[01097] In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In some embodiments, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In some embodiments, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In some embodiments, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In some embodiments, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In some embodiments, the genetically engineered bacterium is killed by the bacterial toxin. In some embodiments, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In some embodiments, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In some embodiments, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the antitoxin is no longer expressed when the exogenous environmental condition is no longer present.

[01098] In some embodiments, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted

heterologous gene encoding a bacterial toxin by the third recombinase.

[01099] In some embodiments, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In some embodiments, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In some embodiments, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In some embodiments, the first excision enzyme excises a first essential gene. In some embodiments, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.

[01100] In some embodiments, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In some embodiments, the wherein the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In some embodiments, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In some embodiments, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In some embodiments, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.

[01101] In some embodiments, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

[01102] In any of these embodiment, the recombinase can be a

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

[01103] In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill- switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. An exemplary kill- switch in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) is shown in Figs. 29-32. The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxin gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arabinose system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

[01104] Thus, in some embodiments, in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an anti-toxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

fir

[01105] In some embodiments, the argA gene is directly or indirectly under the control of the araBAD promoter. Fig. 13 depicts a schematic diagram of an

construct. In this embodiment, the argA

fir

gene is inserted between the araC and araD genes. ArgA is flanked by a ribosome binding site, a FRT site, and one or more transcription terminator sequences. The nucleic acid sequence of an exemplary BAD promoter-driven argA^fbr construct is shown in Table 11. All bolded sequences are Nissle genomic DNA. A portion of the araC gene is bolded and underlined, the argA ¹"^' gene is boxed, and the bolded sequence in between is the promoter that is activated by the presence of arabinose. The ribosome binding site is in italics, the terminator sequences are highlighted, and the FRT site is !>boxed^. A portion of the araD gene is boxed; in dashes. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the BAD promoter sequence of SEQ ID NO: 67 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise the BAD promoter sequence of SEQ ID NO: 67 or a functional fragment thereof.

Table 41

|AGATCGACGCAAATCTGGCTGCGCATCACCACGAACCGCTGTATCACAAGAATATA| CGTGTGACCGACGCCAAAACACTGGAACTGGTGAAGCAGGCTGCGGGAACATTGCAl

ACTGGATATTACTGCTCGCCTGTCGATGAGTCTCAATAACACGCCGCTGCAGGGCG

CGCATATCAACGTCGTCAGTGGCAATTTTATTATTGCCCAGCCGCTGGGCGTCGAT

GACGGCGTGGATTACTGCCATAGCGGGCGTATCCGGCGGATTGATGAAGACGCGAT

CCATCGTCAACTGGACAGCGGTGCAATAGTGCTAATGGGGCCGGTCGCTGTTTCAG

TCACTGGCGAGAGCTTTAACCTGACCTCGGAAGAGATTGCCACTCAACTGGCCATC

AAACTGAAAGCTGAAAAGATGATTGGTTTTTGCTCTTCCCAGGGCGTCACTAATGA

CGACGGTGATATTGTCTCCGAACTTTTCCCTAACGAAGCGCAAGCGCGGGTAGAAG

CCCAGGAAGAGAAAGGCGATTACAACTCCGGTACGGTGCGCTTTTTGCGTGGCGCA

GTGAAAGCCTGCCGCAGCGGCGTGCGTCGCTGTCATTTAATCAGTTATCAGGAAGA

TGGCGCGCTGTTGCAAGAGTTGTTCTCACGCGACGGTATCGGTACGCAGATTGTGA

TGGAAAGCGCCGAGCAGATTCGTCGCGCAACAATCAACGATATTGGCGGTATTCTG

GAGTTGATTCGCCCACTGGAGCAGCAAGGTATTCTGGTACGCCGTTCTCGCGAGCA

GCTGGAGATGGAAATCGACAAATTCACCATTATTCAGCGCGATAACACGACTATTG

CCTGCGCCGCGCTCTATCCGTTCCCGGAAGAGAAGATTGGGGAAATGGCCTGTGTG

GCAGTTCACCCGGATTACCGCAGTTCATCAAGGGGTGAAGTTCTGCTGGAACGCAT

TGCCGCTCAGGCTAAGCAGAGCGGCTTAAGCAAATTGTTTGTGCTGACCACGCGCA

GTATTCACTGGTTCCAGGAACGTGGATTTACCCCAGTGGATATTGATTTACTGCCC

GAGAGCAAAAAGCAGTTGTACAACTACCAGCGTAAATCCAAAGTGTTGATGGCGGA

AGCTCCAGCCTACACAATCGCTCAAGACGTGTAATGCTGCAATCTGCATGCAAGCT TGGCACTGGCCACGCAAAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAATTTGAT GCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCGCA ACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAA ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGAT GCCTGGCAGTTCCCTACTCTCGCATGctcgagccatgggacgtcicaggtattagaai igccaacctggcgctgccaaaacacaacctggtcacgctcacctggggcaatgtcag;cgccgttgatcgcgggcgcggcgtcctggtgatcaaaccttccggcgtcgactaca igcatcatgaccgctgacgatatggtcgtggtcagcatcgaaaccggtgaagtggtt: jgaaggtacgaaaaagccctcctccgacacgccaactcaccggctgctctatcaggc: iattcccgtctattggcggcattgtgcacacacactcgcgccacgccaccatctggg jcgcaggcgggccagtcgattccagcagccggcaccacccacgccgactatttctac;ggcaccattccctgcacccgcaaaatgaccgacgcagaaatcaacggtgaatatga igtgggaaaccggtaacgtcatcgtagaaaccttcgaaaaacagggtatcaatgcag jcgcaaatgcccggcgtgctggtccattctcacggcccatttgcatggggaaaaaac;gccgaagatgcggtgcataacgccatcgtgctggaagaagtcgcttatatggggat iattctgccgtcagttagcgccgcagttaccggatatgcagcaaacgctgctggata; iaacactatctgcgtaagcatggcgcgaaggcatattacgggcagtaa;

[01106] Arabinose inducible promoters are known in the art, including

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

[01107] In one exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding a tetracycline repressor protein (TetR), a Parac promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the tetracycline repressor protein (PTetiO- In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In some embodiments, the araC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.

[01108] In some embodiments, of the disclosure, the recombinant bacterial cell further comprises an anti-toxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the anti-toxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.

[01109] In another embodiment of the disclosure, the recombinant bacterial cell further comprises an anti-toxin under the control of the ParaBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti- toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.

[01110] In another exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contain a kill- switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a Parac promoter operably linked to a heterologous gene encoding the AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill- switch system described directly above. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetPv/toxin/anti-toxin kill-switch system described directly above.

[01111] In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long- lived toxin. In this system, the bacterial cell produces equal amounts of toxin and antitoxin to neutralize the toxin. However, if/when the cell loses the plasmid, the shortlived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer- lived toxin killing it.

[01112] In some embodiments, the engineered bacteria of the present disclosure, for example, bacteria expressing argA^^r and repressor ArgR further comprise the gene(s) encoding the components of any of the above-described kill- switch circuits.

[01113] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, lbs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B 17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin lb, colicin 5, colicinlO, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

[01114] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNAl, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccE^CTD, MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, ImlO, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

[01115] In some embodiments, the bacterial toxin is bactericidal to the genetically engineered bacterium. In some embodiments, the bacterial toxin is bacteriostatic to the genetically engineered bacterium. [01116] In some embodiments, the engineered bacteria provided herein have an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine- mediated repression of each of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct, e.g., citrulline, in the arginine biosynthesis pathway, such that the mutant arginine regulon produces more arginine and/or intermediate byproduct than an unmodified regulon from the same bacterial subtype under the same conditions. In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argA^. In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, arginino succinate synthase, arginino succinate lyase, and carbamoylphosphate synthase, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria further comprise an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by an oxygen level-dependent promoter. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from the fumarate and nitrate reductase regulator (FNR) promoter, arginine deiminiase and nitrate reduction (ANR) promoter, and dissimilatory nitrate respiration regulator (DNR) promoter. In some embodiments, the arginine feedback resistant N- acetylglutamate synthase mutant is argA^ .

[01117] In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon, wherein the bacterium comprises a gene encoding a functional N- acetylglutamate synthetase that is mutated to reduce arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding the mutated N- acetylglutamate synthetase is controlled by a promoter that is induced under low- oxygen or anaerobic conditions, wherein the mutant arginine regulon comprises one or more operons comprising genes that encode arginine biosynthesis enzymes N- acetylglutamate kinase, N-acetylglutamate phosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, carbamoylphosphate synthase, ornithine transcarbamylase, arginino succinate synthase, and arginino succinate lyase, and wherein each operon comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine-mediated repression of the operon via ArgR repressor binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the genes in the operon.

[01118] In some embodiments, the genetically engineered bacteria is an auxotroph comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the genetically engineered bacteria comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[01119] In some embodiments, the genetically engineered bacteria comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant further comprises a kill- switch circuit, such as any of the kill- switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD- In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin.

[01120] In some embodiments, the genetically engineered bacteria is an auxotroph comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.

[01121] In some embodiments, of the above described genetically engineered bacteria, the gene encoding the arginine feedback resistant N- acetylglutamate synthetase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding the arginine feedback resistant N- acetylglutamate synthetase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[01122] In some embodiments, the genetically engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive, or the genetically engineered bacteria do not have an arginine repressor (e.g. , the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria further comprise an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the arginine feedback resistant N- acetylglutamate synthase mutant is controlled by an oxygen level-dependent promoter. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from the fumarate and nitrate reductase regulator (FNR) promoter, arginine deiminiase and nitrate reduction (ANR) promoter, and dissimilatory nitrate respiration regulator (DNR) promoter. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is argA^.

[01123] In some embodiments, the genetically engineered bacteria comprise a mutant or deleted arginine repressor and an arginine feedback resistant N- acetylglutamate synthase mutant. In some embodiments, the genetically engineered bacterium comprise an arginine regulon, wherein the bacterium comprises a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions and wherein the bacterium has been genetically engineered to lack a functional ArgR repressor.

[01124] In some embodiments, the genetically engineered bacteria comprising a mutant or deleted arginine repressor and an arginine feedback resistant N- acetylglutamate synthase mutant is an auxotroph. In some embodiments, the genetically engineered bacteria comprising a mutant or deleted arginine repressor and an arginine feedback resistant N-acetylglutamate synthase mutant is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[01125] In some embodiments, the genetically engineered bacteria comprising a mutant or deleted arginine repressor and an arginine feedback resistant N- acetylglutamate synthase mutant further comprise a kill- switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD- In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

[01126] In some embodiments, the genetically engineered bacterium is an auxotroph comprising a mutant or deleted arginine repressor and an arginine feedback resistant N-acetylglutamate synthase mutant and further comprises a kill- switch circuit, such as any of the kill-switch circuits described herein.

[01127] In some embodiments, of the above described genetically engineered bacteria, the gene encoding the arginine feedback resistant N- acetylglutamate synthetase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding the arginine feedback resistant N- acetylglutamate synthetase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

Transport

[01128] Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell.

[01129] The inner membrane protein YddG of E. coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al, FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over- express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.

[01130] In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria or genetically engineered oncolytic viruses comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria or genetically engineered oncolytic viruses comprise one or more copies of aroP, tnaB or mtr gene.

[01131] In some embodiments, the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP.

[01132] In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transporter gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.

[01133] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[01134] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.

[01135] In some embodiments, the native transporter gene is

mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome.

[01136] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non- native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. [01137] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter.

Pharmaceutical Compositions and Formulations

[01138] Pharmaceutical compositions comprising the genetically engineered bacteria described herein may be used to treat, manage, ameliorate, and/or prevent a disorder associated with hyperammonemia or symptom(s) associated with hyperammonemia. Pharmaceutical compositions comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[01139] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein.

[01140] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's

Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[01141] The genetically engineered bacteria described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g. , liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g. , oral, topical, injectable, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 105 to 1012 bacteria, e.g. , approximately 105 bacteria, approximately 106 bacteria, approximately 107 bacteria, approximately 108 bacteria, approximately 109 bacteria, approximately 1010 bacteria, approximately 1011 bacteria, or approximately 1011 bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In some embodiments, the pharmaceutical composition is administered before the subject eats a meal. In some embodiments, the pharmaceutical composition is administered currently with a meal. In some embodiments, the pharmaceutical composition is administered after the subject eats a meal.

[01142] The composition may be administered once or more daily, weekly, or monthly. The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the

pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g. , 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from

hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[01143] The genetically engineered bacteria disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well- known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g. , osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g. , a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In some embodiments, the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.

[01144] The genetically engineered bacteria disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[01145] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. , pregelatinised maize starch, polyvinylpyrrolidone, hydro xypropyl methylcellulose,

carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g. , lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid,

polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g. , starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate- polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG- A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA- MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly

pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N- dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan- locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly- anhydrides, starch

polymethacrylates, polyamino acids, and enteric coating polymers.

[01146] In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

[01147] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g. , sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g. , lecithin or acacia); non-aqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria described herein.

[01148] In some embodiments, the genetically engineered bacteria of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et ah , 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, In some embodiments, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In some embodiments, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

[01149] In some embodiments, the composition suitable for

administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In some embodiments, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors. [01150] In some embodiments, the composition suitable for

administration to pediatric subjects may include a flavor. As used herein, "flavor" is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

[01151] In certain embodiments, the genetically engineered bacteria may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or coadminister the compound with, a material to prevent its inactivation.

[01152] In another embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product. In some embodiments, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria- fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In some embodiments, the food product is a fermented food, such as a fermented dairy product. In some embodiments, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In some embodiments, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. For example, see U.S. 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.

[01153] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g. , conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[01154] The genetically engineered bacteria described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g.,

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g. , of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[01155] The genetically engineered bacteria may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g. , as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[01156] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g. , by infusion.

[01157] Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[01158] In other embodiments, the composition can be delivered in a controlled release or sustained release system. In some embodiments, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of therapies of the present disclosure (see, e.g., U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly( vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[01159] Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a

predetermined period of time, or the dose may be reduced or increased as indicated by therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

[01160] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[01161] The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In some embodiments, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water- free concentrate in a hermetically sealed container and can be reconstituted (e.g. , with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0- 10% sucrose (optimally 0.5- 1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0- 0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g. , hyaluronidase.

[01162] In some embodiments, the disclosure provides a composition comprising a combination (e.g., two or more) of different genetically engineered bacteria, each bacteria encoding and producing a different effector molecule.

[01163] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more enzymes for the production of tryptophan or one of its metabolites and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more different enzymes for the production of a different tryptophan metabolite.

[01164] In some embodiments, the composition comprises one or more genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the production of a short chain fatty acid.

[01165] In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the production of a butyrate. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the production of acetate. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of an antiinflammatory cytokine. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of IL-10. In some

embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of IL-22. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of IL-2. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of IL-27. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of IL-22. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of GLP2. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of a satiety effector, e.g., GLP2. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the catabolism of branched chain amino acids.

[01166] In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of a proinflammatory cytokine or growth factor or agonist , e.g. IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM- CSF. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion of IL-15. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion or display of an antibody, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PD1, PDL1, CTLA4, anti-LAG3, anti-TIM3). In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion or display of an anti-PDl antibody. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for the secretion or display of an anti-PD-Ll antibody. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria comprising one or more gene sequences for production of arginine. In some embodiments, the composition comprises one or more genetically engineered bacteria comprising one or more gene sequences encoding one or more polypeptides for the production of tryptophan or one or more of its metabolites, and further comprises one or more additional genetically engineered bacteria

comprising one or more gene sequences for the catabolism of adenosine.

[01167] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the production of a gut barrier enhancer molecule, e.g., butyrate, acetate, propionate are described in pending International Patent Application PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety.

[01168] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an ant i- inflammatory effector molecule, e.g., IL10, IL-22, IL-2, IL-27, or a gut barrier enhancer, e.g., GLP2, or a satiety effector, e.g., GLP1, are described in in pending International Patent Application

PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the secretion of an immuno-stimulatory cytokine, e.g., IL-15 and other effectors (IL-12, IL-2, IL-15, IL-18, IL-7, IL-21, CD40 agonist, CD40 agonist, CD226 agonist, CD 137 agonist, ICOS agonist, OXO40 agonist, GM-CSF, antibodies, e.g., scFvs, including but not limited to checkpoint inhibitors (e.g., PDl, PDLl, CTLA4, anti-LAG3, anti-TIM3), or for the production of adenosine, for the production and secretion or display of immune checkpoint inhibitors, e.g., anti-PD-1 or anti-PD-Ll and others, are described in International Patent Application PCT/US2017/013072, the contents of which is herein incorporated by reference in its entirety.

[01169] Non- limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the production of arginine, are described in

International Patent Publication WO2016200614, the contents of which is herein incorporated by reference in its entirety. Non-limiting examples of genetically engineered bacteria and gene sequence(s) useful for the for the catabolism of branched chain amino acids, are described in pending International Patent Publication

WO2016/201380, the contents of which is herein incorporated by reference in its entirety. [01170] The pharmaceutical composition comprising the genetically engineered bacteria may be administered alone or in combination with one or more additional therapeutic agents, e.g., a chemotherapeutic drug such a methotrexate. An important consideration in selecting the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria. In some studies, the efficacy of anticancer immunotherapy, e.g., CTLA-4 or PD-1 inhibitors, requires the presence of particular bacterial strains in the microbiome (Ilda et al., 2013; Vetizou et al., 2015; Sivan et al., 2015). In some embodiments, the pharmaceutical composition is administered with one or more commensal or probiotic bacteria, e.g., Bifidobacterium or Bacteroides.

[01171] In some embodiments, the genetically engineered bacteria are administered sequentially, simultaneously, or subsequently to dosing with one or more chemotherapeutic agents selected from Trabectedin®, Belotecan®, Cisplatin®, Carboplatin ®, Bevacizumab®, Pazopanib®, 5-Fluorouracil, Capecitabine®,

Irinotecan®, and Oxaliplatin®. In some embodiments, the genetically engineered bacteria are administered sequentially, simultaneously, or subsequently to dosing with Gemcitabine (Gemzar).

[01172] In a non-limiting example one or more genetically engineered bacteria comprising gene sequence(s) encoding one or more enzymes for the

degradation of kynurenine circuits described herein are administered sequentially, simultaneously, or subsequently to dosing with one or more chemotherapeutic reagents described herein. In a non- limiting example one or more genetically engineered bacteria comprising gene sequence(s) encoding one or more enzymes for the degradation of kynurenine circuits described herein and one or more tryptophan production circuits described herein, are administered sequentially, simultaneously, or subsequently to dosing with one or more chemotherapeutic reagents described herein.

[01173] In a non-limiting example, one or more engineered bacteria comprising one or more enzymes for the degradation of kynurenine and/or tryptophan production described herein which further comprise gene sequence(s) encoding anti- CD40 antibody for production and secretion or display of anti-CD40 antibody into the extracellular environment, are administered sequentially, simultaneously, or

subsequently to dosing with one or more chemotherapeutic reagents described herein. In a non- limiting example, one or more engineered bacteria described herein which comprise gene sequence(s) encoding one or more one or more enzymes for the degradation of kynurenine and/or tryptophan production and further comprise gene sequence(s) encoding enzymes for the degradation of adenosine, are administered sequentially, simultaneously, or subsequently to dosing with one or more

chemotherapeutic reagents described herein. In these embodiments, the one or more chemotherapeutic reagent is administered systemically and/or orally and/or

intratumorally.

[01174] In a non-limiting example one or more genetically engineered bacteria comprising gene sequence(s) encoding one or more one or more enzymes for the degradation of kynurenine and/or tryptophan production and further comprising gene sequence(s) encoding one or more adenosine degradation enzyme(s) described herein are administered sequentially, simultaneously, or subsequently to dosing with Gemcitabine. In a non-limiting example, one or more engineered bacteria described herein which comprise gene sequence(s) encoding one or more one or more enzymes for the degradation of kynurenine and/or tryptophan production and which further comprise gene sequence(s) encoding anti-CD40 antibody for production and secretion and/or display of anti-CD40 antibody into the extracellular environment, are

administered sequentially, simultaneously, or subsequently to dosing with Gemcitabine. In these embodiments, Gemcitabine is administered systemically and/or orally and/or intratumorally.

[01175] In some embodiments, the one or more genetically engineered bacteria is administered sequentially, simultaneously, or subsequently to dosing with one or more of the following checkpoint inhibitors or other antibodies known in the art or described herein. Non- limiting examples include CTLA-4 antibodies (including but not limited to Ipilimumab and Tremelimumab (CP675206)), anti-4-lBB (CD137, TNFRSF9) antibodies (including but not limited to PF-05082566, and Urelumab), anti CD 134 (OX40) antibodies, including but not limited to Anti-OX40 antibody

(Providence Health and Services), anti-PDl antibodies (including but not limited to Nivolumab, Pidilizumab, Pembrolizumab (MK-3475/SCH900475, lambrolizumab, REGN2810, PD1 (Agenus)), anti-PD-Ll antibodies (including but not limited to Durvalumab (MEDI4736), Avelumab (MSB0010718C), and Atezolizumab

(MPDL3280A, RG7446, R05541267)), andit-KIR antibodies (including but not limited to Lirilumab), LAG3 antibodies (including but not limited to BMS-986016), anti-CCR4 antibodies (including but not limited to Mogamulizumab), anti-CD27 antibodies (including but not limited to Varlilumab), anti- CXCR4 antibodies (including but not limited to Ulocuplumab). In some embodiments, the at least one bacterial cell is administered sequentially, simultaneously, or subsequently to dosing with an anti- phophatidyl serine antibody (including but not limited to Bavituxumab).

[01176] In some embodiments, the one or more bacteria is administered sequentially, simultaneously, or subsequently to dosing with one or more antibodies selected from TLR9 antibody (including, but not limited to, MGN1703 PD1 antibody (including, but not limited to, SHR-1210 (Incyte/Jiangsu Hengrui)), anti-OX40 antibody (including, but not limited to, OX40 (Agenus)), anti-Tim3 antibody (including, but not limited to, Anti-Tim3 (Agenus/INcyte)), anti-Lag3 antibody (including, but not limited to, Anti-Lag3 (Agenus/INcyte)), anti-B7H3 antibody (including, but not limited to, Enoblituzumab (MGA-271), anti- CT-011 (hBAT, hBATl) as described in

WO2009101611, anti-PDL-2 antibody (including, but not limited to, AMP-224

(described in WO2010027827 and WO2011066342)), anti-CD40 antibody (including, but not limited to, CP-870, 893), anti-CD40 antibody (including, but not limited to, CP- 870, 893).

[01177] In a non-limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more one or more enzymes for the degradation of kynurenine and/or tryptophan production, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PDl antibody. In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more one or more enzymes for the degradation of kynurenine and/or tryptophan production and further comprise gene sequence(s) encoding enzymes for the production of arginine, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PDl antibody. Non- limiting examples of such anti-PDl antibodies are described herein. In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more one or more enzymes for the degradation of kynurenine and/or tryptophan production and further comprise gene sequence(s) encoding anti-CD47 antibody for production and secretion or display of anti-CD47 antibody into the extracellular environment and optionally further comprise gene sequence(s) encoding enzymes for the production of arginine, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PDl antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of advanced solid tumors. In some embodiments, the genetically engineered bacteria for the treatment of advanced solid tumors are administered orally. In some embodiments, the genetically engineered bacteria for the treatment of advanced solid tumors are administered systemically. In some embodiments, the genetically engineered bacteria for the treatment of advanced solid tumors are administered intratumorally. In these embodiments, the anti-PD-1 antibody is administered systemically and/or orally and/or intratumorally.

[01178] In a non-limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or tryptophan production further comprise gene sequence(s) encoding one or more cytokine(s) described herein for production and secretion of one or more cytokine(s) into the extracellular environment, are

administered sequentially, simultaneously, or subsequently to dosing with an anti-PDl antibody. Non- limiting examples of such anti-PD-Ll and/or PD-1 antibodies are described herein, and include but are not limited to, Keytruda (pembrolizumab, anti-PD- 1), Optivo (nivolumab, anti-PDl), and Tecentriq (Atezolizumab, anti-PD-Ll). In these embodiments, the anti-PD-1 antibody is administered systemically and/or orally and/or intratumorally. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine and/or the production of tryptophan for the degradation of kynurenine and/or secretes one or more cytokine(s) described herein, alone or in combination with a PD-1 antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of colorectal carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine and/or the production of tryptophan for the degradation of kynurenine and/or secretes one or more cytokine(s) described herein, alone or in combination with a PD-1 antibody, e.g., nivolumab and/or pembrolizumab, is used for the treatment, management and/or prevention of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of hepatocellular carcinoma. In one

embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of hepatocellular carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine and/or the production of tryptophan for the degradation of kynurenine and/or secretes one or more cytokine(s) described herein, alone or in combination with a PD-1 antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of immunotherapy-refractory advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of advanced melanoma.

[01179] In a non-limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or tryptophan production further comprise gene sequence(s) encoding IL-15 for production and secretion of IL-15 into the extracellular environment, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PDl antibody. In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding IL-15 for production and display of IL-15 on the cell surface facing into the extracellular environment in combination with gene sequence(s) encoding encoding one or more enzymes for the degradation of kynurenine, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PDl antibody. Non- limiting examples of such anti- PD-L1 and/or PD-1 antibodies are described herein, and include but are not limited to, Keytruda (pembrolizumab, anti-PD-1), Optivo (nivolumab, anti-PDl), and Tecentriq (Atezolizumab, anti-PD-Ll). In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine for the degradation of kynurenine and/or secretes IL-15, alone or in combination with a PD-1 antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of colorectal carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine for the degradation of kynurenine and/or secretes IL-15, alone or in combination with a PD-1 antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of

hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of hepatocellular carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine for the degradation of kynurenine and/or secretes IL-15, alone or in combination with a PD- 1 antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of immunotherapy-refractory advanced melanoma. In one

embodiment, the administration of the genetically engineered bacterium is oral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of advanced melanoma. In these embodiments, the anti-PD-1 antibody is administered systemically and/or orally and/or intratumorally.

[01180] In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or tryptophan production further comprise gene sequence(s) encoding anti-CD47 antibody for production and secretion or display of anti-CD47 antibody into the extracellular environment, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PD-Ll antibody. In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or tryptophan production further comprise gene sequence(s) encoding enzymes for the production of arginine, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PD-Ll antibody. Non-limiting examples of such anti-PDLl antibodies are described herein. In a non-limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or tryptophan production further comprise gene sequence(s) encoding anti-CD47 antibody for production and secretion or display of anti-CD47 antibody into the extracellular environment in combination with gene sequence(s) encoding enzymes for the production of arginine, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PD-Ll antibody. Non-limiting examples of such anti-PDLl antibodies are described herein. In one embodiment, such a regimen comprising a genetically engineered bacterium which comprises circuitry for the production of arginine and/or for the secretion of anti-CD47, alone or in combination with a PD-L1 antibody, is used for the treatment, management and/or prevention of advanced solid tumors. In some embodiments, the genetically engineered bacteria for the treatment of advanced solid tumors are administered orally. In some embodiments, the genetically engineered bacteria for the treatment of advanced solid tumors are administered systemically. In some embodiments, the genetically engineered bacteria for the treatment of advanced solid tumors are administered intratumorally. In these embodiments, the antiPD-Ll antibody is administered systemically and/or orally and/or intratumorally. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine and/or the production of tryptophan for the degradation of kynurenine and/or secretes one or more cytokine(s) described herein, alone or in combination with a PD-L1 antibody, is used for the treatment, management and/or prevention of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of colorectal carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine and/or the production of tryptophan for the degradation of kynurenine and/or secretes one or more cytokine(s) described herein, alone or in combination with a PD-L1 antibody, e.g., is used for the treatment, management and/or prevention of

hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of hepatocellular carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine and/or the production of tryptophan for the degradation of kynurenine and/or secretes one or more cytokine(s) described herein, alone or in combination with a PD-L1 antibody is used for the treatment, management and/or prevention of immunotherapy- refractory advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of advanced melanoma.

[01181] In a non- limiting example, one or more engineered bacteria described herein, which comprise comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or tryptophan production further gene sequence(s) encoding one or more cytokine(s) described herein for production and secretion of one or more cytokine(s) into the extracellular environment, are

administered sequentially, simultaneously, or subsequently to dosing with an anti-PD- Ll antibody. In a non-limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or the production of tryptophan, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PD-Ll antibody. Non- limiting examples of such anti-PDl antibodies are described herein. In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more cytokine(s) described herein for production and secretion of one or more cytokine(s) into the extracellular environment in combination with gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or the production of tryptophan, are administered sequentially, simultaneously, or subsequently to dosing with an anti-PD-Ll antibody. Non-limiting examples of such anti-PD-Ll and/or PD-1 antibodies are described herein, and include but are not limited to, Keytruda (pembrolizumab, anti-PD-1), Optivo (nivolumab, anti-PDl), and Tecentriq (Atezolizumab, anti-PD-Ll). In these embodiments, the anti-PD-Ll antibody is administered systemically and/or orally and/or intratumorally.

[01182] In a non- limiting example, one or more engineered bacteria described herein, which comprise gene sequence(s) encoding one or more enzymes for the degradation of kynurenine and/or the production of tryptophan and further comprise gene sequence(s) encoding IL-15 for production and secretion of IL-15 into the extracellular environment, are administered sequentially, simultaneously, or

subsequently to dosing with an anti-PD-Ll antibody. Non-limiting examples of such anti-PD-Ll and/or PD-1 antibodies are described herein, and include but are not limited to, Keytruda (pembrolizumab, anti-PD-1), Optivo (nivolumab, anti-PDl), and Tecentriq (Atezolizumab, anti-PD-Ll). In one embodiment, such a regimen comprising a genetically

[01183] engineered bacterium which produces one or more enzymes for the degradation of kynurenine for the degradation of kynurenine and/or secretes IL-15, alone or in combination with a PD-L1 antibody, e.g., nivolumab and/or pebrolizumab, is used for the treatment, management and/or prevention of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of colorectal carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of colorectal carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine for the degradation of kynurenine and/or secretes IL-15, alone or in combination with a PD-L1 antibody, is used for the treatment, management and/or prevention of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is systemic for the treatment of hepatocellular carcinoma. In one embodiment, the administration of the genetically engineered bacterium is intratumoral for the treatment of hepatocellular carcinoma. In one embodiment, such a regimen comprising a genetically engineered bacterium which produces one or more enzymes for the degradation of kynurenine for the degradation of kynurenine and/or secretes IL-15, alone or in combination with a PD-L1 antibody, is used for the treatment, management and/or prevention of immunotherapy-refractory advanced melanoma. In one embodiment, the administration of the genetically engineered bacterium is oral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacteria is intratumoral for the treatment of advanced melanoma. In one embodiment, the administration of the genetically engineered bacteria is systemic for the treatment of advanced melanoma. In these embodiments, the antiPD-Ll antibody is administered systemically and/or orally and/or intratumorally.

[01184] In some embodiments, the PD-L1 antibodies administered in combination with the genetically engineered bacteria are administered systemically. In some embodiments, the PD-1 antibodies administered in combination with the genetically engineered bacteria are administered are administered systemically. In some embodiments, the PD-1 antibodies administered in combination with the genetically engineered bacteria are administered are administered intratumorally. In some embodiments, the PD-L1 antibodies administered in combination with the genetically engineered bacteria are administered are administered intratumorally. In some embodiments, the PD-1 antibodies administered in combination with the genetically engineered bacteria are administered are administered orally. In some embodiments, the PD-L1 antibodies are administered orally.

[01185] In some embodiments, the genetically engineered bacteria are administered systemically. In some embodiments, the genetically engineered bacteria are administered intratumorally. In some embodiments, the genetically engineered bacteria are administered orally.

[01186] In some embodiments, the genetically engineered bacteria are administered intratumorally and the PD-1 antibodies are administered systemically. In some embodiments, the genetically engineered bacteria are administered intratumorally and the PD-Ll antibodies are administered systemically. In some embodiments, the genetically engineered bacteria are administered intratumorally and the PD-1 antibodies are administered intratumorally. In some embodiments, the genetically engineered bacteria are administered intratumorally and the PD-Ll antibodies are administered intratumorally. In some embodiments, the genetically engineered bacteria are administered intratumorally and the PD- 1 antibodies are administered orally. In some embodiments, the PD-Ll antibodies are administered orally.

[01187] In some embodiments, the genetically engineered bacteria are administered systemically and the PD-1 antibodies are administered systemically. In some embodiments, the genetically engineered bacteria are administered systemically and the PD-Ll antibodies are administered systemically. In some embodiments, the th genetically engineered bacteria are administered systemically and PD-1 antibodies are administered intratumorally. In some embodiments, the genetically engineered bacteria are administered systemically and the PD-Ll antibodies are administered

intratumorally. In some embodiments, the genetically engineered bacteria are administered systemically and the PD-1 antibodies are administered orally. In some embodiments, the genetically engineered bacteria are administered systemically and the PD-Ll antibodies are administered orally.

[01188] In some embodiments, the genetically engineered bacteria are administered orally and the PD-1 antibodies are administered systemically. In some embodiments, the genetically engineered bacteria are administered and orally the PD-Ll antibodies are administered systemically. In some embodiments, the genetically engineered bacteria are administered orally and the PD- 1 antibodies are administered intratumorally. In some embodiments, the genetically engineered bacteria are administered orally and the PD-Ll antibodies are administered intratumorally. In some embodiments, the genetically engineered bacteria are administered orally and the PD-1 antibodies are administered orally. In some embodiments, the genetically engineered bacteria are administered orally and the PD-Ll antibodies are administered orally.

[01189] In some embodiments, the genetically engineered

microorganisms may be administered as part of a regimen, which includes other treatment modalities or combinations of other modalities. Non-limiting examples of these modalities or agents are conventional therapies (e.g., radiotherapy, chemotherapy), other immunotherapies, stem cell therapies, and targeted therapies, (e.g., BRAF or vascular endothelial growth factor inhibitors; antibodies or compounds), bacteria described herein, and oncolytic viruses. Therapies also include related to antibody- immune engagement, including Fc-mediated ADCC therapies, therapies using bispecific soluble scFvs linking cytotoxic T cells to tumor cells (e.g., BiTE), and soluble TCRs with effector functions. Immunotherapies include vaccines (e.g., viral antigen, tumor associated antigen, neoantigen, or combinations thereof), checkpoint inhibitors, cytokine therapies, adoptive cellular therapy (ACT). ACT includes but is not limited to, tumor infiltrating lymphocyte (TIL) therapies, native or engineered TCR or CAR-T therapies, natural killer cell therapies, and dendritic cell vaccines or other vaccines of other antigen presenting cells. Targeted therapies include antibodies and chemical compounds, and include for example antiangiogenic strategies and BRAF inhibition.

[01190] In one embodiment, the genetically engineered microorganism is an oncolytic virus. In some embodiments, the genetically engineered OV is delivered in combination with vaccines, chemotherapy, radiotherapy, checkpoint inhibitors, chemoradiotherapy, anti-angiogenic therapy, monoclonal antibodies, adoptive cell transfer, cytokines, chemokines, other OVs and any of the modalities mentioned above.

[01191] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more enzymes for the degradation of kynurenine and/or for the production of tryptophan are administered in combination with a short chain fatty acid, e.g., butyrate.

Methods of Treatment

[01192] Another aspect of the invention provides methods of treating a disease or disorder associated with pathogenic up or downregulation of the immune system. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases or disorders. In some embodiments, the disorder is any cancer as described herein, HIV, viral hepatitis, herpes virus, parasitic infection, multiple sclerosis, rheumatoid arthritis, systemic lupus erythrmatosis, psioriasis, graft vs. host disease, IBD, colitis, colorectal cancer, Huntington's disease, Alzheimer's disease, Parkinson's disease, amylotrophic lateral sclerosis, seizure disorders, depressive disorders, attention deficit disorder, autism, schizophrenia, chronic inflammatory syndrome, pain, schizophrenia, chronic brain injury, metabolic syndrome, obesisty, diabetes type I and II, insulin resistance, non-alcoholic seatohepatitis, hepatic encephalopathy, Artherosclerosis, chronic kidney failure, hypoxia, ischeimia, stroke, sepsis, pellagra, hepatic porphyria, bone

morphogenesis.

[01193] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally, e.g. , in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g. , by enema. In some embodiments, the genetically engineered bacteria of the invention are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically.

[01194] In certain embodiments, administering the pharmaceutical composition to the subject reduces or increases tryptophan or its metabolites in a subject. In some embodiments, the methods of the present disclosure may reduce or increase tryptophan or its metabolite concentrations in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing the ammonia concentration in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating the condition allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[01195] Before, during, and after the administration of the pharmaceutical composition, ammonia concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the invention to reduce or increases tryptophan or its metabolites in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's ammonia concentrations prior to treatment.

[01196] In certain embodiments, the genetically engineered bacteria E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al, 2009), or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition may be re- administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice is shown in Figs. 14 and 15. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

[01197] The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents. An important

consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria.

[01198] In some embodiments, the genetically engineered bacteria are administered for prevention, treatment or management of any of the diseases described herein. In some embodiments, the genetically engineered bacteria are administered in combination with another therapeutic approach to prevent reoccurrence of any of the diseases described herein. In some embodiments, the genetically engineered bacteria are administered in combination with branched-chain amino acid supplementation. In some embodiments, the genetically engineered bacteria are administered in combination with one or more checkpoint inhibitors.

[01199] In some embodiments, the genetically engineered bacteria are administered in combination with one or more antibiotics, for example for the treatment of HE. Examples of such antibiotics include, but are not limited to, non-absorbable antibiotics, such as aminoglicosides, e.g., neomycin and/or paramomycin. In some embodiments, the antibiotic is rifamycin. In some embodiments, the antibiotic is a rifamycin derivative, e.g., a synthetic derivative, including but not limited to, rifaximin.

[01200] In any of the enbodiments described herein, for any of the diseases described herein, the genetically engineered bacteria are administered in combination with any of the conventional or experimental therapies known in the art for the treatment of the disease. [01201] In some embodiments, the pharmaceutical composition is administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food. The pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g. , low-protein diet and amino acid supplementation. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

Treatment In Vivo

[0100] The genetically engineered bacteria of the invention may be evaluated in vivo, e.g. , in an animal model. Any suitable animal model of a disease or condition may be used as described herein.

Screening Methods

[01202] In some embodiments, of the disclosure a genetically engineered strain may be improved upon by using screening and selection methods, e.g. , to increase PME enzymatic activity or to increase the ability of a strain to take up phenylalanine. In some embodiments, the screen serves to generate a bacterial strain with improved PME activity. In some embodiments, the screen serves to generate a bacterial strain which has improved phenylalanine uptake ability. In some embodiments, the screen may identify a bacterial strain with both improved PME activity and enhanced substrate import. Non- limiting examples of methods of screening which can be used are described herein.

Generation of Bacterial Strains with Enhance Ability to Transport Biomolecules

[01203] Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population. [01204] This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite.

[01205] For example, if the biosynthetic pathway for producing an amino acid is disrupted a strain capable of high-affinity capture of said amino acid can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the amino acid at regular intervals. Over time, cells that are most competitive for the amino acid - at growth-limiting concentrations - will come to dominate the population. These strains will likely have mutations in their amino acid-transporters resulting in increased ability to import the essential and limiting amino acid.

[01206] Similarly, by using an auxotroph that cannot use an upstream metabolite to form an amino acid, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways.

[01207] In the previous examples, a metabolite innate to the microbe was made essential via mutational auxo trophy and selection was applied with growth- limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate. Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

[01208] Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE

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

[01209] Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations "screened" throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 10 11 2 CCD 1. This rate can be accelerated by the addition of chemical mutagens to the cultures - such as N-methyl-N-nitro-N-nitrosoguanidine (NTG) - which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

[01210] At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvo luted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. 0. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

[01211] In some embodiments, the ALE method can be used to identify genetically engineered bacteria with improved phenylalanine uptake.

Optimization of Kynurenine consumption and Tryptophan Production

(ALE)

[01212] E. coli Nissle can be engineered to efficiently import KYN and convert it to TRP. While Nissle does not typically utilize KYN, by introducing the Kynureninase (KYNase) from Pseudomonas fluorescens (kynU) on a medium-copy plasmid under the control of the tetracycline promoter (Ptet) a new strain with this plasmid (Ptet-KYNase) is able to convert L-kynurenine into anthranilate. E. coli naturally utilizes anthranilate in its TRP biosynthetic pathway. Briefly, the TrpE (in complex with TrpD) enzyme converts chorismate into anthranilate. TrpD, TrpC, TrpA and TrpB then catalyzes a five-step reaction ending with the condensation of an indole with serine to form tryptophan. By replacing the TrpE enzyme via lambda- RED recombineering, the subsequent strain of Nissle (AtrpE::Cm) is an auxotroph unable to grow in minimal media without supplementation of TRP or anthranilate. By expressing kynureninase in AtrpE .Cm (KYNase-trpE), this auxotrophy can be alternatively rescued by providing KYN.

[01213] Leveraging the growth-limiting nature of KYN in KYNase-trpE, adaptive laboratory evolution was employed to further evolve a strain capable of increasingly efficient utilization of KYN. First a lower limit of KYN concentration was established and mutants were evolved by passaging in lowering concentrations of KYN. While this can select for mutants capable of increasing KYN import, the bacterial cells still prefer to utilize free, exogenous TRP. In the tumor environment, dual-therapeutic functions can be provided by depletion of KYN and increasing local concentrations of TRP. Therefore, to evolve a strain which prefers KYN over TRP, a toxic analogue of TRP - 5-fluoro-L-tryptophan (ToxTRP) - can be incorporated into the ALE experiment. The resulting best performing strain is then whole genome sequenced in order to deconvolute the contributing mutations. Lambda-RED can be performed in order to reintroduce TrpE, to inactivate Trp regulation (trpR, tyrR, transcriptional attenuators) to up-regulate TrpABCDE expression and increase chorismate production. The resulting strain is now insensitive to external TRP, efficiently converts KYN into TRP, and also now overproduces TRP.

[01214] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 3B or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 3B or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 3B or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 3B or a functional fragment thereof

[01215] In some embodiments, the genetically engineered bacteria encode a gene or gene cassette, which promotes anti- inflammatory activity. In some

embodiments, the genetically engineered bacteria are capable of producing kynurenine.

[01216] In some embodiments, this step involves the conversion of tryptophan to kynurenine, and may be catalyzed by the ubiquitously-expressed enzyme indoleamine 2,3-dioxygenase (IDO-1), or by tryptophan dioxygenase (TDO), an enzyme which is primarily localized to the liver (Alvarado et al, 2015). The

genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise one or more gene(s) or gene cassette(s) for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some embodiments, the genetically engineered bacteria comprise a gene encoding kynurenine formamidase. [01217] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments, the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.

[01218] In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g. , to enhance stability, increase kynurenine production, and/or increase ant i- inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate

transaminase. In some embodiments,

[01219] The genetically engineered bacteria may comprise any suitable gene for producing kynurenic acid. In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g. , to enhance stability, increase kynurenic acid production, and/or increase ant i- inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g. , under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions.

[01220] In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYN ratio, e.g. in the circulation. In some embodimetns the TRP:KYN ratio is increased. In some embodiments, TRP:KYN ratio is decreased, some embodiments, the genetically engineered bacteria the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the KYNA:QUIN ratio.

[01221] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut or the tumor micorenvironment, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g. , high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g. , thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Examples

[0101] The following examples provide illustrative embodiments, of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.

Example 1. Transforming E. coli

[0102] Each plasmid is transformed into E. coli Nissle or E. coli DH5a. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli Nissle or E. coli DH5a is diluted 1 : 100 in 5 mL of lysogeny broth (LB) and grown until it reached an OD600 of 0.4-0.6. The cell culture medium contains a selection marker, e.g. , ampicillin, that is suitable for the plasmid. The E. coli cells are then centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are finally resuspended in 0.1 mL of 4° C water. The

electroporator is set to 2.5 kV. 0.5 μg of one of the above plasmids is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. One mL of room- temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing ampicillin and incubated overnight.

[01 03] In alternate embodiments, the tryptophan cassette can be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, MA). Organization of the constructs and nucleotide sequences are provided herein. To create a vector capable of integrating the synthesized butyrate cassette construct into the chromosome, Gibson assembly is first used to add lOOObp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly is used to clone the fragment between these arms. PCR is used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the tryptophan cassette between them. This PCR fragment is used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature- sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells are grown out for 2 hours before plating on chloramphenicol at 20ug/mL at 37 degrees C. Growth at 37 degrees C also cures the pKD46 plasmid.

Transformants containing cassette are chloramphenicol resistant and lac-minus (lac-).

Example 2. Verifying mutants

[0104] The presence of the mutation is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 μΐ of cold dd¾0 by pipetting up and down. 3 μΐ_^ of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C overnight. A PCR master mix is made using 5 μΐ. of 10X PCR buffer, 0.6 μΐ of 10 mM dNTPs, 0.4 μΐ. of 50 mM Mg₂S0₄, 6.0 μϊ_^ of 10X enhancer, and 3.0 μΐ_^ of ddH₂0 (15 μΐ_^ of master mix per PCR reaction). A 10 μΜ primer mix is made by mixing 2 μϊ_^ of primers unique to the tryptophan mutant construct (100 μΜ stock) into 16 μΐ_^ of ddH₂0. For each 20 μΐ_^ reaction, 15μί of the PCR master mix, 2.0 μΐ_^ of the colony suspension (template), 2.0 μΐ_^ of the primer mix, and 1.0 μΐ_^ of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C at 5:00 min., 2) 94° C at 0: 15 min., 3) 55° C at 0:30 min., 4) 68° C at 2:00 min., 5) 68° C at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 μΐ_^ of each amplicon and 2.5 μΐ_^ 5X dye. The PCR product only forms if the mutation has inserted into the genome.

Example 3. Removing selection marker

[0105] The antibiotic resistance gene is removed with pCP20. Each strain with the mutation is grown in LB media containing antibiotics at 37° C until it reaches an OD₆oo of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 niL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pCP20 plasmid DNA is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette was placed into the sample chamber, and the electric pulse was applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1-3 hrs. The cells are spread out on an LB plate containing kanamycin and incubated overnight. Colonies that do not grow to a sufficient OD₆oo overnight are further incubated for an additional 24 hrs. 200 μΐ_^ of cells are spread on ampicillin plates, 200 μΐ_^ of cells are spread on kanamycin plates, and both are grown at 37° C overnight. The ampicillin plate contains cells with pCP20. The kanamycin plate provides an indication of how many cells survived the electroporation. Transformants from the ampicillin plate are purified no n- selectively at 43° C and allowed to grow overnight.

Example 4. Engineering bacterial strains using chromosomal insertions

[0106] Bacterial strains, in which the kyreninase constructs are integrated directly into the E. coli Nissle genome under the control of an FNR-responsive promoter, are constructed.

[0107] To create a vector capable of integrating the PfnrS- kyreninase construct into the chromosome at the Nissle lacZ locus, Gibson assembly is used to add 1000 bp sequences of DNA homologous to the Nissle lacZ locus to both sides of a flippase recombination target (FRT) site-flanked chloramphenicol resistance (cmR) cassette on a knock-in knock-out (KIKO) plasmid. Gibson assembly is then used to clone the PfnrS- kyreninase construct DNA sequence between these homology arms, adjacent to the FRT-cmR-FRT site. Successful insertion of the fragment is validated by sequencing. PCR is used to amplify the entire lacZ::FRT-cmR-FRT:: PfnrS- kyreninase construct: :lacZ region. This knock- in PCR fragment is used to transform an

electrocompetent Nissle strain that contains a temperature- sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells are grown for 2 hrs at 37 °C. Growth at 37 °C cures the temperature- sensitive plasmid. Transformants with successful chromosomal integration of the fragment are selected on chloramphenicol at 20 μg/mL.

[0108] To create a vector capable of integrating the PfnrS- kyreninase construct into the E. coli Nissle chromosome at Nissle malP and malT loci, Gibson assembly is used to add 1000 bp sequences of DNA homologous to the Nissle malP and malT loci on either side of an FRT site-flanked kanamycin resistance (knR) cassette on a KIKO plasmid. Gibson assembly is then used to clone the PfnrS- kyreninase y DNA sequence between these homology arms, adjacent to the FRT-knR-FRT site. Successful insertion of the fragment is validated by sequencing. PCR is used to amplify the entire malP::FRT-knR-FRT:: PfnrS- kyreninase construct: :malT region. This knock- in PCR fragment is used to transform an electrocompetent Nissle strain already containing PfnrS- kyreninase construct in the lacZ locus, and expressing the lambda red

recombinase genes. After transformation, cells are grown for 2 hrs at 37 °C. Transformants with successful integration of the fragment are selected on kanamycin at 50 μg/mL. These same methods may be used to create a vector capable of integrating the PfnrS- kyreninase sequence at the malE/K insertion site.

[0109] In some embodiments, recombinase-based switches may be used to activate kyreninase construct expression. To construct a strain allowing recombinase- based switches to regulate kyreninase expression, the PfnrS-driven Int5 gene and the rrnBUP-driven, recombinase site-flanked kyureninase sequences are synthesized by Genewiz (Cambridge, MA). Gibson assembly is used to add 1000 bp sequences of DNA homologous to the Nissle malP and malT loci on either side of the PfnrS-Int5, rrnBUP- kyureninase DNA sequence and to clone this sequence between the homology arms. Successful insertion of the fragment into a KIKO plasmid is validated by sequencing. PCR is used to amplify the entire PfnrS-Int5, rrnBUP- kyreninase region. This knock- in PCR fragment is used to transform an electrocompetent Nissle strain expressing the lambda red recombinase genes. After transformation, cells are grown for 2 hrs at 37 °C. Transformants with successful integration of the PfnrS- kyreninase fragment at the malPT intergenic region are selected on kanamycin at 50 μg/mL. This strategy may also be used to construct a recombinase-based strain requiring T7 polymerase activity for kyureninase expression.

Example 6. Detection and quantification of Kynurenine

[0110] Secreted KP metabolites are detectable systemically in plasma or serum, and cerebrospinal fluid (CSF), or alternatively can be detected in in vitro-derived cell culture supernatants. Sensitive methods of separation, such as high pressure liquid chromatography (HPLC) coupled with gas chromatography/mass spectrometry

(GC/MS) have been used to detect KP metabolites independent of their biological source (Chen, et ah, Characterization of the kynurenine pathway in NSC-34 cell line: implications for amyotrophic lateral sclerosis. J Neurochem 118(5): 816-825; Lovelace, et ah, Recent evidence for an expanded role of the Kynurenine pathway of tryptophan metabolism in neurological diseases, Neuropharmacology (2016), and references therein). The kynurenine to tryptophan ratio (KYN/TRP) is a routinely used, suitable indicator of IDO-1 activity, and thus of TRP degradation. The ratio represents the concentration of the product of IDO-1 and TDO (kynurenine) versus the concentration of TRP, their substrate (Schrocksnadel, Wirleitner et al. 2006). [0111] A kynurenine assay is described as follows (see Forrest et al, Purine,

Kynurenine, Neopterin and Lipid Peroxidation Levels in Inflammatory Bowel Disease; J Biomed Sci 2002;9:436-442)):

[0112] A 480-microliter sample of the serum is used for the assay, to which 20

II of 3-nitro-L-tyrosine solution were added as internal standard. To this mixture, 50 microliter of 3.3 M perchloric acid is added, then the sample is vortex mixed and centrifuged at 13,000 g for 10 min. Sample preparation and centrifugation is performed at 4° C. The supernatant is injected onto a Waters gradient HPLC system, using a C18 reverse-phase column and a mobile phase with the following composition: solvent A: 0.1% trifluoro acetic acid; solvent B: a 3: 1 mixture of acetonitrile and water containing 0.1% trifluoro acetic acid. The gradient used for separation started with 90% A and 10% B, changing to 77% A and 23% B by 20 min. The kynurenines are detected using a UV detector at 254 nm.

Example 9. Kynurenine production in recombinant E. coli

[0113] Production of kynurenine is assessed in E. coli Nissle strains containing the kynurenine cassette comprising IDO described above in order to determine the effect of oxygen on kynurenine production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% C02, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for kynurenine concentration confirm that kynurenine production in these recombinant strains can be achieved in a low-oxygen environment.

[0114] In other embodiments, other conditions, and other inducible promoters, such as ROS and RNS are tested. Example 10. Administration of C. novyi expressing human anti-PD-1 antibody, IL- 15, and kynureninase (depleting kynurenin) in humanized mice as a model for colon cancer

[0115] Humanized CD34+ mice (NOD scid gamma mice engrafted with

CD34+ cells through injection of CD34+ hematopoietic stem cells) are purchased from Jackson Labs (Pearson et ah, 2008), bred, and maintained under specific pathogen- free conditions.

[0116] HT-29 cell line is obtained from ATCC and cultured according to the guidelines provided. Approximately 3 X 10⁶ tumor cells are implanted on the flank of Humanized CD34+ mice. Animals are randomized based on tumor size and treatment is initiated when tumors reach 100 mm .

[0117] For i.v. injection and intratumoral injection, bacteria are grown in LB broth until reaching an absorbance at 600 nm (A600 nm) of 0.4 (corresponding to 2 X 10 colony-forming units (CFU)/mL) and washed twice in PBS. The suspension is then diluted to so that 100 microL can be injected at the appropriate doses into the lateral tail vein or intratumorally into tumor-bearing mice. Bacterial spores are suspended in 0.1 ml of PBS for injection. Vehicle control mice are injected with 0.1 mL PBS via tail vein.

[0118] For intratumoral administration, mice are administered either C. novyi expressing human anti-PD-1 antibody, IL-15, and kynureninase, or the control bacterial spores (wild-type C. novyi). A control group is injected with the same volume of PBS. Intratumoral injections are performed 2 to 3 times weekly with various doses of bacteria expressing a anti- human PD-1 antibody or control bacteria, including the optimum dose determined as described above.

[0119] For intravenous administration, mice are intravenously administered either C. novyi expressing human anti-PD-1 antibody, IL-15, and kynureninase, or the control bacteria (wild-type C. novyi) through tail vein injection at doses ranging from 1

X 10 5^J to 1 X 108°. A control group is injected with the same volume of PBS.

[0120] For oral administration, mice are gavaged with 5 X 10⁹ CFU bacteria, either C. novyi expressing human anti-PD-1 antibody, IL-15, and kynureninase, or the control bacteria (wild-type C. novyi), as described in Danino et ah, 2015. [0121] Tumors are measured two to three times per week until study termination. Tumor Volume is calculated (length 9 (width 9 width) 9 0.5) = volume in mm³.

Example 11. Administration of E.Coli nissle expressing human anti-CTLA-4 antibody, IL-12, and capable of producing tryptophan in a humanized mouse tumor model for breast cancer

[0122] Humanized CD34+ mice (NOD scid gamma mice engrafted with

CD34+ cells through injection of CD34+ hematopoietic stem cells) are purchased from Jackson Labs (Pearson et al., 2008), bred, and maintained under specific pathogen- free conditions.

[0123] EMT-6 (breast cancer derived) cell line is obtained from ATCC and cultured according to guidelines provided. Approximately 3 X 10⁶ tumor cells are implanted on the flank of humanized CD34+ mice. When the tumor reaches 100 mm3, animals are randomized and treatment is initiated.

[0124] For i.v. injection and intratumoral injection, bacteria are grown in LB broth until reaching an absorbance at 600 nm (A600 nm) of 0.4 (corresponding to 2 X 10 colony-forming units (CFU)/mL) and washed twice in PBS. The suspension is then diluted to so that 100 microL can be injected at the appropriate doses into the lateral tail vein or intratumorally into tumor-bearing mice. Bacteria suspended in 0.1 ml of PBS for injection. Vehicle control mice are injected with 0.1 mL PBS via tail vein.

[0125] For oral administration, bacteria are prepared by growth in LB until exponential phase, washed three times with sterile PBS, and then diluted to the appropriate concentration in sterile PBS.

[0126] For intratumoral administration, mice are administered either E.Coli nissle expressing human anti-CTLA-4 antibody, IL-12, and a tryptophan cassette for the production of tryptophan or the control bacteria (wild-type E. Coli nissle). A control group is injected with the same volume of PBS. Intratumoral injections are performed 2 to 3 times weekly with various doses of bacteria expressing ipilimumab or control bacteria, including the optimum dose determined as described above.

[0127] For intravenous administration, mice are intravenously administered either E.Coli nissle expressing human anti-CTLA-4 antibody, IL-12, and a tryptophan cassette for the production of tryptophan or the control bacteria (wild-type E. Coli nissle) through tail vein injection at doses ranging from 1 X 10⁵ to 1 X 10⁹. A control group is injected with the same volume of PBS.

[0128] For oral administration, mice are gavaged with 5 X 10⁹ CFU E.Coli nissle expressing human anti-CTLA-4 antibody, IL-12, and a tryptophan cassette for the production of tryptophan or the control bacteria (wild-type E. Coli nissle), as described in Danino et ah, 2015

[0129] Tumors are measured two to three times per week until study termination.

Example 12. Generation of E.Coli Mutants with Ability to Consume L-Kynurenine and Produce Tryptophan from Kynurenine

[0130] E. coli Nissle can be engineered to efficiently import KYN and convert it to TRP. A strain was constructed with a knock out in TrpE (tryptophan auxotroph) that also expresses exogenous Pseudomonas fluorescens kynureninase on a medium copy plasmid and dirven by a tetracycline inducible promoter (Table 42). In the presence of tetracycline, this strain is capable of converting L-kynurenine to

anthranilate. Anthranilate can then be converted tryptophan through the enzymes of the tryptophan biosynthetic pathway.

Table 42. Constructs for Tet-inducible Expression of Pseudomonas fluorescens

Kynureninase

CCTGTATATTGCGGAAGGGTTGGCGGATATGCT

GCAACAAGGTTACACTCTGCGTTTGGTGGATTC

ACCGGAAGAGCTGCCACAGGCTATAGATCAGG

ACACCGCGGTGGTGATGCTGACGCACGTAAATT

ATAAAACCGGTTATATGCACGACATGCAGGCTC

TGACCGCGTTGAGCCACGAGTGTGGGGCTCTGG

CGATTTGGGATCTGGCGCACTCTGCTGGCGCTG

TGCCGGTGGACCTGCACCAAGCGGGCGCGGAC

TATGCGATTGGCTGCACGTACAAATACCTGAAT

GGCGGCCCGGGTTCGCAAGCGTTTGTTTGGGTT

TCGCCGCAACTGTGCGACCTGGTACCGCAGCCG

CTGTCTGGTTGGTTCGGCCATAGTCGCCAATTC

GCGATGGAGCCGCGCTACGAACCTTCTAACGGC

ATTGCTCGCTATCTGTGCGGCACTCAGCCTATT

ACTAGCTTGGCTATGGTGGAGTGCGGCCTGGAT

GTGTTTGCGCAGACGGATATGGCTTCGCTGCGC

CGTAAAAGTCTGGCGCTGACTGATCTGTTCATC

GAGCTGGTTGAACAACGCTGCGCTGCACACGA

ACTGACCCTGGTTACTCCACGTGAACACGCGAA

ACGCGGCTCTCACGTGTCTTTTGAACACCCCGA

GGGTTACGCTGTTATTCAAGCTCTGATTGATCG

TGGCGTGATCGGCGATTACCGTGAGCCACGTAT

TATGCGTTTCGGTTTCACTCCTCTGTATACTACT

TTTACGGAAGTTTGGGATGCAGTACAAATCCTG

GGCGAAATCCTGGATCGTAAGACTTGGGCGCA

GGCTCAGTTTCAGGTGCGCCACTCTGTTACTTA

A

Pseudomonas TAATTCCTAATTTTTGTTGACACTCTATCATTGA fluorescens TAGAGTTATTTTACCACTCCCTATCAGTGATAG kynureninase, codon AGAAAAGTGAATTATATAAAAGTGGGAGGTGC optimized for CCGAATGACGACCCGAAATGATTGCCTAGCGTT expression in E. coli GGATGCACAGGACAGTCTGGCTCCGCTGCGCCA driven by a Tet ACAATTTGCGCTGCCGGAGGGTGTGATATACCT inducible promoter GGATGGCAATTCGCTGGGCGCACGTCCGGTAGC

TGCGCTGGCTCGCGCGCAGGCTGTGATCGCAGA

SEQ ID NO: 153 AGAATGGGGCAACGGGTTGATCCGTTCATGGA

ACTCTGCGGGCTGGCGTGATCTGTCTGAACGCC

TGGGTAATCGCCTGGCTACCCTGATTGGTGCGC

GCGATGGGGAAGTAGTTGTTACTGATACCACCT

CGATTAATCTGTTTAAAGTGCTGTCAGCGGCGC

TGCGCGTGCAAGCTACCCGTAGCCCGGAGCGCC

GTGTTATCGTGACTGAGACCTCGAATTTCCCGA

CCGACCTGTATATTGCGGAAGGGTTGGCGGATA

TGCTGCAACAAGGTTACACTCTGCGTTTGGTGG

ATTCACCGGAAGAGCTGCCACAGGCTATAGATC

AGGACACCGCGGTGGTGATGCTGACGCACGTA

AATTATAAAACCGGTTATATGCACGACATGCAG

GCTCTGACCGCGTTGAGCCACGAGTGTGGGGCT

CTGGCGATTTGGGATCTGGCGCACTCTGCTGGC GCTGTGCCGGTGGACCTGCACCAAGCGGGCGC

GGACTATGCGATTGGCTGCACGTACAAATACCT

GAATGGCGGCCCGGGTTCGCAAGCGTTTGTTTG

GGTTTCGCCGCAACTGTGCGACCTGGTACCGCA

GCCGCTGTCTGGTTGGTTCGGCCATAGTCGCCA

ATTCGCGATGGAGCCGCGCTACGAACCTTCTAA

CGGCATTGCTCGCTATCTGTGCGGCACTCAGCC

TATTACTAGCTTGGCTATGGTGGAGTGCGGCCT

GGATGTGTTTGCGCAGACGGATATGGCTTCGCT

GCGCCGTAAAAGTCTGGCGCTGACTGATCTGTT

CATCGAGCTGGTTGAACAACGCTGCGCTGCACA

CGAACTGACCCTGGTTACTCCACGTGAACACGC

GAAACGCGGCTCTCACGTGTCTTTTGAACACCC

CGAGGGTTACGCTGTTATTCAAGCTCTGATTGA

TCGTGGCGTGATCGGCGATTACCGTGAGCCACG

TATTATGCGTTTCGGTTTCACTCCTCTGTATACT

ACTTTTACGGAAGTTTGGGATGCAGTACAAATC

CTGGGCGAAATCCTGGATCGTAAGACTTGGGCG

CAGGCTCAGTTTCAGGTGCGCCACTCTGTTACT

TAA

Pseudomonas TTAAGACCCACTTTCACATTTAAGTTGTTTTTCT fluorescens AATCCGCATATGATCAATTCAAGGCCGAATAAG kynureninase, codon AAGGCTGGCTCTGCACCTTGGTGATCAAATAAT optimized for TCGATAGCTTGTCGTAATAATGGCGGCATACTA expression in E. coli TCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGA (no underline, no CTTGATGCTCTTGATCTTCCAATACGCAACCTA italic) driven by a Tet AAGTAAAATGCCCCACAGCGCTGAGTGCATATA inducible promoter ATGCATTCTCTAGTGAAAAACCTTGTTGGCATA (underlined italic) AAAAGGCTAATTGATTTTCGAGAGTTTCATACT with RBS (italic , and GTTTTTCTGTAGGCCGTGTACCTAAATGTACTTT tetR in reverse TGCTCCATCGCGATGACTTAGTAAAGCACATCT orientation AAAACTTTTAGCGTTATTACGTAAAAAATCTTG (underlined) CCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGT

ATGGTGCCTATCTAACATCTCAATGGCTAAGGC

SEQ ID NO: 154 GTCGAGCAAAGCCCGCTTATTTTTTACATGCCA

ATACAATGTAGGCTGCTCTACACCTAGCTTCTG

GGCGAGTTTACGGGTTGTTAAACCTTCGATTCC

GACCTCATTAAGCAGCTCTAATGCGCTGTTAAT

CACTTTACTTTTATCTAATCTAGACATCAT AAJT

TTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAA

TTATATAAAAGTGGGAGGTGCCCGA ATGACGACC

CGAAATGATTGCCTAGCGTTGGATGCACAGGAC

AGTCTGGCTCCGCTGCGCCAACAATTTGCGCTG

CCGGAGGGTGTGATATACCTGGATGGCAATTCG

CTGGGCGCACGTCCGGTAGCTGCGCTGGCTCGC

GCGCAGGCTGTGATCGCAGAAGAATGGGGCAA

CGGGTTGATCCGTTCATGGAACTCTGCGGGCTG

GCGTGATCTGTCTGAACGCCTGGGTAATCGCCT GGCTACCCTGATTGGTGCGCGCGATGGGGAAGT

AGTTGTTACTGATACCACCTCGATTAATCTGTTT

AAAGTGCTGTCAGCGGCGCTGCGCGTGCAAGCT

ACCCGTAGCCCGGAGCGCCGTGTTATCGTGACT

GAGACCTCGAATTTCCCGACCGACCTGTATATT

GCGGAAGGGTTGGCGGATATGCTGCAACAAGG

TTACACTCTGCGTTTGGTGGATTCACCGGAAGA

GCTGCCACAGGCTATAGATCAGGACACCGCGGT

GGTGATGCTGACGCACGTAAATTATAAAACCGG

TTATATGCACGACATGCAGGCTCTGACCGCGTT

GAGCCACGAGTGTGGGGCTCTGGCGATTTGGGA

TCTGGCGCACTCTGCTGGCGCTGTGCCGGTGGA

CCTGCACCAAGCGGGCGCGGACTATGCGATTGG

CTGCACGTACAAATACCTGAATGGCGGCCCGGG

TTCGCAAGCGTTTGTTTGGGTTTCGCCGCAACT

GTGCGACCTGGTACCGCAGCCGCTGTCTGGTTG

GTTCGGCCATAGTCGCCAATTCGCGATGGAGCC

GCGCTACGAACCTTCTAACGGCATTGCTCGCTA

TCTGTGCGGCACTCAGCCTATTACTAGCTTGGC

TATGGTGGAGTGCGGCCTGGATGTGTTTGCGCA

GACGGATATGGCTTCGCTGCGCCGTAAAAGTCT

GGCGCTGACTGATCTGTTCATCGAGCTGGTTGA

ACAACGCTGCGCTGCACACGAACTGACCCTGGT

TACTCCACGTGAACACGCGAAACGCGGCTCTCA

CGTGTCTTTTGAACACCCCGAGGGTTACGCTGT

TATTCAAGCTCTGATTGATCGTGGCGTGATCGG

CGATTACCGTGAGCCACGTATTATGCGTTTCGG

TTTCACTCCTCTGTATACTACTTTTACGGAAGTT

TGGGATGCAGTACAAATCCTGGGCGAAATCCTG

GATCGTAAGACTTGGGCGCAGGCTCAGTTTCAG

GTGCGCCACTCTGTTACTTAAGGAG 131] In other embodiments, human kynureninase is used (Table 43).

Table 43 Constructs for the Expression of Human Kynureninase

GTTAATCTGCACCTGCTGATGCTGTCTTTTTTTAAACCG

ACCCCGAAACGCTACAAAATACTGCTGGAAGCGAAAG

CGTTTCCGTCGGATCACTATGCTATAGAAAGTCAACTG

CAGTTGCATGGTCTGAATATCGAGGAATCTATGCGCAT

GATTAAACCGCGTGAGGGTGAAGAAACGCTGCGTATT

GAAGACATTCTGGAAGTTATTGAAAAAGAAGGTGATT

CTATCGCAGTTATACTGTTTTCTGGCGTGCACTTTTATA

CAGGTCAGCACTTCAATATCCCGGCAATCACTAAAGCG

GGGCAGGCAAAAGGCTGCTATGTTGGTTTTGACCTGGC

GCATGCAGTGGGGAATGTTGAACTGTATCTGCACGATT

GGGGCGTTGATTTCGCGTGTTGGTGTAGCTACAAATAT

CTGAACGCTGGCGCGGGTGGCATTGCTGGCGCTTTTAT

TCACGAAAAACACGCGCACACCATTAAACCGGCTCTG

GTTGGCTGGTTCGGTCATGAGCTGAGTACTCGCTTTAA

AATGGATAACAAACTGCAATTGATTCCGGGTGTTTGCG

GCTTCCGTATCAGCAATCCGCCGATTCTGCTGGTTTGC

AGCCTGCACGCTAGTCTGGAAATCTTTAAGCAGGCGAC

TATGAAAGCGCTGCGCAAAAAATCTGTGCTGCTGACCG

GCTATCTGGAGTATCTGATCAAACACAATTATGGCAAA

GATAAAGCTGCAACTAAAAAACCGGTAGTGAACATTA

TCACCCCCTCACACGTGGAGGAGCGCGGTTGTCAGCTG

ACTATTACTTTCAGTGTACCTAATAAAGATGTGTTCCA

GGAACTGGAAAAACGCGGCGTTGTTTGTGATAAACGT

AACCCGAATGGTATTCGCGTGGCTCCTGTGCCGCTGTA

CAATTCATTCCACGATGTTTATAAATTCACCAACCTGC

TGACTTCTATTCTCGACAGTGCTGAGACTAAAAATTAA

Human TAATTCCTAATTTTTGTTGACACTCTATCATTGATAGAG kynureninase, TTATTTTACCACTCCCTATCAGTGATAGAGAAAAGTGA codon optimized ATATCAAGACACGAGGAGGTAAGATTATGGAGCCTTC for expression in ATCTTTAGAACTGCCAGCGGACACGGTGCAGCGCATCG E. coli driven by CGGCGGAACTGAAGTGCCATCCGACTGATGAGCGTGT a Tet inducible GGCGCTGCATCTGGACGAAGAAGATAAACTGCGCCAC promoter TTTCGTGAATGTTTTTATATTCCTAAAATTCAAGACTTG

CCGCCGGTAGATTTGAGTCTCGTTAACAAAGATGAAAA

SEQ ID NO: CGCGATCTACTTTCTGGGCAACTCTCTGGGTCTGCAAC 156 CAAAAATGGTTAAAACGTACCTGGAGGAAGAACTGGA

TAAATGGGCAAAAATCGCGGCTTATGGTCACGAAGTG

GGCAAGCGTCCTTGGATTACTGGCGACGAGTCTATTGT

GGGTTTGATGAAAGATATTGTGGGCGCGAATGAAAAG

GAAATTGCACTGATGAATGCTCTGACCGTTAATCTGCA

CCTGCTGATGCTGTCTTTTTTTAAACCGACCCCGAAAC

GCTACAAAATACTGCTGGAAGCGAAAGCGTTTCCGTCG

GATCACTATGCTATAGAAAGTCAACTGCAGTTGCATGG

TCTGAATATCGAGGAATCTATGCGCATGATTAAACCGC

GTGAGGGTGAAGAAACGCTGCGTATTGAAGACATTCT

GGAAGTTATTGAAAAAGAAGGTGATTCTATCGCAGTTA

TACTGTTTTCTGGCGTGCACTTTTATACAGGTCAGCACT

TCAATATCCCGGCAATCACTAAAGCGGGGCAGGCAAA

AGGCTGCTATGTTGGTTTTGACCTGGCGCATGCAGTGG GGAATGTTGAACTGTATCTGCACGATTGGGGCGTTGAT

TTCGCGTGTTGGTGTAGCTACAAATATCTGAACGCTGG

CGCGGGTGGCATTGCTGGCGCTTTTATTCACGAAAAAC

ACGCGCACACCATTAAACCGGCTCTGGTTGGCTGGTTC

GGTCATGAGCTGAGTACTCGCTTTAAAATGGATAACAA

ACTGCAATTGATTCCGGGTGTTTGCGGCTTCCGTATCA

GCAATCCGCCGATTCTGCTGGTTTGCAGCCTGCACGCT

AGTCTGGAAATCTTTAAGCAGGCGACTATGAAAGCGCT

GCGCAAAAAATCTGTGCTGCTGACCGGCTATCTGGAGT

ATCTGATCAAACACAATTATGGCAAAGATAAAGCTGC

AACTAAAAAACCGGTAGTGAACATTATCACCCCCTCAC

ACGTGGAGGAGCGCGGTTGTCAGCTGACTATTACTTTC

AGTGTACCTAATAAAGATGTGTTCCAGGAACTGGAAA

AACGCGGCGTTGTTTGTGATAAACGTAACCCGAATGGT

ATTCGCGTGGCTCCTGTGCCGCTGTACAATTCATTCCA

CGATGTTTATAAATTCACCAACCTGCTGACTTCTATTCT

CGACAGTGCTGAGACTAAAAATTAA

Human TAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCG kynureninase CATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCT codon optimized CTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGT for expression in AATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCT E. coli (no TTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATAC underline, no GCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCA italic) driven by TATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAA a Tet inducible AAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTC promoter TGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGC (underlined GATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTA italic) with RBS TTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGG (italic , and tetR GCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGG in reverse CTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGC orientation CAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGC (underlined) GAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCAT

TAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTAT

SEQ ID NO: CT AATCT AG AC ATC ΑΊ ΑΑ TTCCTAA TTTTTGTTGA CA CTC 157 TATCATTGATAGAGTTATTTTACCACTCCCTATCAGTGATAG

A GAAAA GTGAA TA TCAA GA CACGA GGA GGTAA GA JTATGG

AGCCTTCATCTTTAGAACTGCCAGCGGACACGGTGCAG

CGCATCGCGGCGGAACTGAAGTGCCATCCGACTGATG

AGCGTGTGGCGCTGCATCTGGACGAAGAAGATAAACT

GCGCCACTTTCGTGAATGTTTTTATATTCCTAAAATTCA

AGACTTGCCGCCGGTAGATTTGAGTCTCGTTAACAAAG

ATGAAAACGCGATCTACTTTCTGGGCAACTCTCTGGGT

CTGCAACCAAAAATGGTTAAAACGTACCTGGAGGAAG

AACTGGATAAATGGGCAAAAATCGCGGCTTATGGTCA

CGAAGTGGGCAAGCGTCCTTGGATTACTGGCGACGAG

TCTATTGTGGGTTTGATGAAAGATATTGTGGGCGCGAA

TGAAAAGGAAATTGCACTGATGAATGCTCTGACCGTTA

ATCTGCACCTGCTGATGCTGTCTTTTTTTAAACCGACCC

CGAAACGCTACAAAATACTGCTGGAAGCGAAAGCGTT TCCGTCGGATCACTATGCTATAGAAAGTCAACTGCAGT

TGCATGGTCTGAATATCGAGGAATCTATGCGCATGATT

AAACCGCGTGAGGGTGAAGAAACGCTGCGTATTGAAG

ACATTCTGGAAGTTATTGAAAAAGAAGGTGATTCTATC

GCAGTTATACTGTTTTCTGGCGTGCACTTTTATACAGGT

CAGCACTTCAATATCCCGGCAATCACTAAAGCGGGGC

AGGCAAAAGGCTGCTATGTTGGTTTTGACCTGGCGCAT

GCAGTGGGGAATGTTGAACTGTATCTGCACGATTGGGG

CGTTGATTTCGCGTGTTGGTGTAGCTACAAATATCTGA

ACGCTGGCGCGGGTGGCATTGCTGGCGCTTTTATTCAC

GAAAAACACGCGCACACCATTAAACCGGCTCTGGTTG

GCTGGTTCGGTCATGAGCTGAGTACTCGCTTTAAAATG

GATAACAAACTGCAATTGATTCCGGGTGTTTGCGGCTT

CCGTATCAGCAATCCGCCGATTCTGCTGGTTTGCAGCC

TGCACGCTAGTCTGGAAATCTTTAAGCAGGCGACTATG

AAAGCGCTGCGCAAAAAATCTGTGCTGCTGACCGGCT

ATCTGGAGTATCTGATCAAACACAATTATGGCAAAGAT

AAAGCTGCAACTAAAAAACCGGTAGTGAACATTATCA

CCCCCTCACACGTGGAGGAGCGCGGTTGTCAGCTGACT

ATTACTTTCAGTGTACCTAATAAAGATGTGTTCCAGGA

ACTGGAAAAACGCGGCGTTGTTTGTGATAAACGTAACC

CGAATGGTATTCGCGTGGCTCCTGTGCCGCTGTACAAT

TCATTCCACGATGTTTATAAATTCACCAACCTGCTGAC

TTCTATTCTCGACAGTGCTGAGACTAAAAATTAA

[0132] E. coli naturally utilizes anthranilate in its TRP bio synthetic pathway.

Briefly, the TrpE (in complex with TrpD) enzyme converts chorismate into anthranilate. TrpD, TrpC, TrpA and TrpB then catalyze a five- step reaction ending with the condensation of an indole with serine to form tryptophan. Next, the kynureninase is introduced into a strain which harbors AtrpE (trypophan auxotrophy) deletion. By deleting the TrpE enzyme via lambda-RED recombineering, the subsequent strain of Nissle (AtrpE .Cm) is an auxotroph unable to grow in minimal media without supplementation of TRP or anthranilate. By expressing kynureninase in AtrpE .Cm (KYNase-trpE), this auxotrophy should alternatively rescued by providing KYN.

[0133] Indeed, as a proof of concept, we showed that -while Nissle does not typically utilize KYN - by introducing the kynureninase (KYNase) from Pseudomonas fluorescens (kynU) on a medium-copy plasmid under the control of the tetracycline promoter (Ptet) a new strain with this plasmid (Ptet-KYNase) was able to convert L- kynurenine into anthranilate in the presence of a Tet inducer.

Table 44. STRAIN Rich Media Min Media Min + Min +

Anthranilate KYNU+ aTc

Wild type + + + +

Nissle

trpE + - + - trpE + - + +

pseudoKYNase

trpE hKYNase + - + -

[0134] In a preliminary assay (Table 15), wildtype Nissle (SYN094), Nissle with a deletion of trpE, and trpE mutants expressing either the human kynureninase (hKYNase) or the Pseudomonas fluorescens kynureninase (pseudoKYNase) from a Ptet promoter on a medium-copy plasmid were grown in either rich media, minimal media (min media), minimal media with 5 mM anthranilate (Min + anthranilate) or minimal media with 10 mM kynurenine and 100 ng/uL aTc (Min + KYNU + aTc). These were grown in 1 mL of media in a deep well plate with shaking at 37°C. A positive for growth (+) in Table 15 indicates a change in optical density of >5-fold from inoculation.

[0135] The results show that in a mutant trpE (which is typically used in the tryptophan bio synthetic pathway to convert chorismate into anthranilate) background, Nissle is unable to grow in minimal media without supplementation with anthranilate (or tryptophan). When minimal media was supplemented with KYNU, the trpE mutant was also unable to grow. However, when the Pseudomonas KYNase was expressed in the trpE tryptophan-auxotroph the cells were able to grow in Min+KYNU. This indicates that Nissle is able to import L-kynurenine from the media and convert it into anthranilate using the pseudoKYNase. The human KYNase homolog was unable to support growth on M9+KYNU, most likely due to differences in substrate specificity as it has been documented that the human kynureninase prefers 3-hydroxykynurenine as a substrate (Phillips, Structure and mechanism of kynureninase. Arch Biochem Biophys. 2014 Feb 15;544:69-74).

[0136] Together these experiments establish that expression of the

Pseudomonas fluorescens kynureninase is sufficient to rescue a trpE auxotrophy in the presence of kynurenine, as the strain ia able to consume KYN into anthranilate, and upstream metabolite in the TRP biosynthetic pathway. In addition, the KYNase is also capable of providing increased resistance to the toxic tryptophan, 5-fluoro-L- tryptophan. Using the information attained here it is possible to proceed to an adapative laboratory evolution experiment to select for mutants with highly efficient and selective conversion of kynurenine to tryptophan.

Example 13. Generation of E. Coli Mutants with enhanced ability to consume L- Kynurenine and produce tryptophan from Kynurenine

[0137] Adaptive Laboratory Evolution was used to produce mutant bacterial strains with improved kynurenine consumption and reduced tryptophan uptake.

[0138] Prior to evolving the strains, a lower limit of kynurenine (KYN) concentration was established for use in the ALE experiment.

[0139] While lowering the KYN concentration can select for mutants capable of increasing KYN utilization, the bacterial cells still prefer to utilize free, exogenous TRP. In the tumor environment, dual-therapeutic functions can be provided by depletion of KYN and increasing local concentrations of TRP. Therefore, to evolve a strain which prefers KYN over TRP, a toxic analogue of TRP - 5-fluoro-L-tryptophan (ToxTRP) - can be incorporated into the ALE experiment.

[0140] A checkerboard growth assay was performed in 96-well plates using streptomycin resistant Nissle, deltatrpE and deltatrpE pseudoKYNase with and without induction of pseudoKYNase expression using 100 ng/uL aTc. Detailed procedures used for the checkerboard assay are described in Example 14. Strains were inoculated at very dilute concentrations into M9 minimal media with varying concentrations of KYN across columns (2-fold dilutions starting at 2000 ug/mL) and varying concentrations of ToxTrp across rows (2-fold dilutions starting at 200 ug/mL). On a separate plate, the strains were grown in M9+KYN (at the same concentrations) in the absence of ToxTrp.

[0141] The results of the initial checkerboard assay are shown in FIG. 10,

FIG. 11, and FIG. 12 as a function of optical density at 600 nm (normalized to a media blank). In FIG. 10 and FIG. 11, the X-axis shows decreasing KYNU concentration from left-to-right, while the Z-axis shows decreasing ToxTrp concentration from front- to-back with the very back row representing media with no ToxTrp. In FIG. 10, the controls and trpE strains are shown in M9+KYNU without any ToxTrp, as there was no growth detected from either strain at any concentration of ToxTrp. The results of the assay show that expression of the pseudoKYNase provides protection against toxicity of ToxTrp. More importantly, growth is permitted between 250-62.5 ug/mL of KYNU and 6.3-1.55 ug/mL of ToxTrp.

Example 14. Checkerboard Assay and ALE Parameters

[0142] To establish the minimum concentration of L-kynurenine and maximum concentration of 5-fluoro-L-tryptophan (ToxTrp) capable of sustaining growth of the KYNase strain, using a checkerboard assay, the following protocol was used. Using a 96-well plate with M9 minimal media with glucose, KYN was supplemented decreasing across columns in 2-fold dilutions from 2000 ug/mL down to ~1 ug/mL. In the rows, ToxTrp concentration decreased by 2-fold from 200 ug/mL down to -1.5 ug/mL. In one plate, Anhydrous Tetracycline (aTc) was added to a final concentration of 100 ng/uL to induce production of the KYNase. From an overnight culture, cells were diluted to an OD600 = 0.5 in 12 mL of TB (plus appropriate antibiotics and inducers, where applicable) and grown for 4 hours. 100 uL of cells were spun down and resuspended to an OD600 = 1.0. These were diluted 2000-fold and 25 uL was added to each well to bring the final volumes in each well to 100 uL. Cells were grown for roughly 20 hours with static incubation at 37C then growth was assessed by OD600, making sure readings fell within linear range (.05-1.0).

Example 15. Determination of ALE Parameters

[0143] Once identified, the highest concentrations of ToxTrp and lowest concentration of kynurenine capable of supporting growth becomes the starting point for ALE. The ALE parental strain was chosen by culturing the KYNase strain on M9 minimal media supplemented with glucose and L-kynurenine (referred to as M9+KYN from here on). A single colony was selected, resuspended in 20 uL of sterile phosphate- buffered saline solution. This colony was then used to inoculate three cultures of M9+KYN, grown into late-logarithmic phase and optical density determined at 600 nm. These cultures were then diluted to 10 in 4 rows of a 96-well deep-well plate with 1 mL of M9+KYN. Each one of the four rows has a different ToxTrp (increasing 2-fold), while each column has decreasing concentrations of KYN (by 2-fold). Each morning and evening this plate is diluted back to 10 using the well in which the culture has grown to just below saturation so that the culture is always in logarithmic growth. This process is repeated until a change in growth rate is no longer detected. Once no growth rate increases are detected (usually around 10¹¹ Cumulative Cell Divisions) the culture is plated onto M9+KYN (Lee, et ah, Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172; 2011). Individual colonies are selected and screened in

M9+KYN+ToxTrp media to confirm increased growth rate phenotype. Once mutants with significantly increased growth rate on M9+KYN are isolated, genomic DNA can be isolated and sent for whole genome sequencing to reveal the mutations responsible for phenotype. All culturing is done shaking at 350 RPM at 37°C.

[0144] The resulting best performing strain can them be whole genome sequenced in order to deconvolute the contributing mutations. In some embodiments, Lambda-RED can be performed in order to reintroduce TrpE, to inactivate Trp regulation (trpR, tyrR, transcriptional attenuators) to up-regulate TrpABCDE expression and increase chorismate production. The resulting strain prefers external KYN over to external TRP, efficiently converts KYN into TRP, and also now overproduces TRP.

Example 16. ALE

[0145] First, strains were generated, which comprise the trpE knock out and integrated constructs for the expression of Pseudomonas fluorescens KYNase driven by a constitutive promoter (Table 45). KYNase constructs were integrated at the HA3/4 site, and two different promoters were used; the promoter of the endogenous lpp gene was used in parental strain SYN2027 (HA3/4::Plpp-pKYNase KanR TrpE::CmR) and the synthetic pSynJ23119 was used in parental strain SYN2028 (HA3/4::PSynJ23119- pKYNase KanR TrpE::CmR). These strains were generated so that a strain would be evolved, which would comprise a chromosomally integrated version of Pseudomonas fluorescens KYNase.

Table 45. Constructs for Constitutive Expression of Pseudomonas fluorescens

Kynureninase

SEQ ID NO: 159

SYN23119 promoter GGAAAATTTTTTTAAAAAAAAAACTTGACAGCT with RBS AGCTCAGTCCTTGGTATAATGCTAGCACGAAGT

GAATTATATAAAAGTGGGAGGTGCCCGA

SEQ ID NO: 160

Pseudomonas GGAAAATTTTTTTAAAAAAAAAACTTGACAGCT fluorescens, codon AGCTCAGTCCTTGGTATAATGCTAGCACGAAGT optimized for GAATTATATAAAAGTGGGAGGTGCCCGAATGA expression in E. coli, CGACCCGAAATGATTGCCTAGCGTTGGATGCAC driven by the AGGACAGTCTGGCTCCGCTGCGCCAACAATTTG SYN23119 CGCTGCCGGAGGGTGTGATATACCTGGATGGCA

ATTCGCTGGGCGCACGTCCGGTAGCTGCGCTGG

Construct can be CTCGCGCGCAGGCTGTGATCGCAGAAGAATGG expressed from a GGCAACGGGTTGATCCGTTCATGGAACTCTGCG plasmid, e.g., pl5 or GGCTGGCGTGATCTGTCTGAACGCCTGGGTAAT can be integrated into CGCCTGGCTACCCTGATTGGTGCGCGCGATGGG the chromosome, GAAGTAGTTGTTACTGATACCACCTCGATTAAT e.g., at the HA3/4 site CTGTTTAAAGTGCTGTCAGCGGCGCTGCGCGTG

CAAGCTACCCGTAGCCCGGAGCGCCGTGTTATC

SEQ ID NO: 161 GTGACTGAGACCTCGAATTTCCCGACCGACCTG

TATATTGCGGAAGGGTTGGCGGATATGCTGCAA

CAAGGTTACACTCTGCGTTTGGTGGATTCACCG

GAAGAGCTGCCACAGGCTATAGATCAGGACAC

CGCGGTGGTGATGCTGACGCACGTAAATTATAA

AACCGGTTATATGCACGACATGCAGGCTCTGAC

CGCGTTGAGCCACGAGTGTGGGGCTCTGGCGAT

TTGGGATCTGGCGCACTCTGCTGGCGCTGTGCC

GGTGGACCTGCACCAAGCGGGCGCGGACTATG

CGATTGGCTGCACGTACAAATACCTGAATGGCG

GCCCGGGTTCGCAAGCGTTTGTTTGGGTTTCGC

CGCAACTGTGCGACCTGGTACCGCAGCCGCTGT

CTGGTTGGTTCGGCCATAGTCGCCAATTCGCGA

TGGAGCCGCGCTACGAACCTTCTAACGGCATTG

CTCGCTATCTGTGCGGCACTCAGCCT ATT ACTA

GCTTGGCTATGGTGGAGTGCGGCCTGGATGTGT

TTGCGCAGACGGATATGGCTTCGCTGCGCCGTA

AAAGTCTGGCGCTGACTGATCTGTTCATCGAGC

TGGTTGAACAACGCTGCGCTGCACACGAACTGA

CCCTGGTTACTCCACGTGAACACGCGAAACGCG

GCTCTCACGTGTCTTTTGAACACCCCGAGGGTT

ACGCTGTTATTCAAGCTCTGATTGATCGTGGCG

TGATCGGCGATTACCGTGAGCCACGTATTATGC

GTTTCGGT

Lpp promoter from ATAAGTGCCTTCCCATCAAAAAAATATTCTCAA E. coli CATAAAAAACTTTGTGTAATACTTGTAACGCTA

SEQ ID NO: 162

RBS TTATATAAAAGTGGGAGGTGCCCGA SEQ ID NO: 163

Lpp promoter from ATAAGTGCCTTCCCATCAAAAAAATATTCTCAA E. coli CATAAAAAACTTTGTGTAATACTTGTAACGCTA

GTGAATTATATAAAAGTGGGAGGTGCCCGA

SEQ ID NO: 164

Pseudomonas ATAAGTGCCTTCCCATCAAAAAAATATTCTCAA fluorescens CATAAAAAACTTTGTGTAATACTTGTAACGCTA kynureninase driven GTGAATTATATAAAAGTGGGAGGTGCCCGAAT by GACGACCCGAAATGATTGCCTAGCGTTGGATGC

Lpp promoter from ACAGGACAGTCTGGCTCCGCTGCGCCAACAATT E. coli TGCGCTGCCGGAGGGTGTGATATACCTGGATGG

CAATTCGCTGGGCGCACGTCCGGTAGCTGCGCT

Construct can be GGCTCGCGCGCAGGCTGTGATCGCAGAAGAAT expressed from a GGGGCAACGGGTTGATCCGTTCATGGAACTCTG plasmid, e.g., pl5 or CGGGCTGGCGTGATCTGTCTGAACGCCTGGGTA can be integrated into ATCGCCTGGCTACCCTGATTGGTGCGCGCGATG the chromosome, GGGAAGTAGTTGTTACTGATACCACCTCGATTA e.g., at the HA3/4 site ATCTGTTTAAAGTGCTGTCAGCGGCGCTGCGCG

TGCAAGCTACCCGTAGCCCGGAGCGCCGTGTTA

SEQ ID NO: 165 TCGTGACTGAGACCTCGAATTTCCCGACCGACC

TGTATATTGCGGAAGGGTTGGCGGATATGCTGC

AACAAGGTTACACTCTGCGTTTGGTGGATTCAC

CGGAAGAGCTGCCACAGGCTATAGATCAGGAC

ACCGCGGTGGTGATGCTGACGCACGTAAATTAT

AAAACCGGTTATATGCACGACATGCAGGCTCTG

ACCGCGTTGAGCCACGAGTGTGGGGCTCTGGCG

ATTTGGGATCTGGCGCACTCTGCTGGCGCTGTG

CCGGTGGACCTGCACCAAGCGGGCGCGGACTA

TGCGATTGGCTGCACGTACAAATACCTGAATGG

CGGCCCGGGTTCGCAAGCGTTTGTTTGGGTTTC

GCCGCAACTGTGCGACCTGGTACCGCAGCCGCT

GTCTGGTTGGTTCGGCCATAGTCGCCAATTCGC

GATGGAGCCGCGCTACGAACCTTCTAACGGCAT

TGCTCGCTATCTGTGCGGCACTCAGCCTATTACT

AGCTTGGCTATGGTGGAGTGCGGCCTGGATGTG

TTTGCGCAGACGGATATGGCTTCGCTGCGCCGT

AAAAGTCTGGCGCTGACTGATCTGTTCATCGAG

CTGGTTGAACAACGCTGCGCTGCACACGAACTG

ACCCTGGTTACTCCACGTGAACACGCGAAACGC

GGCTCTCACGTGTCTTTTGAACACCCCGAGGGT

TACGCTGTTATTCAAGCTCTGATTGATCGTGGC

GTGATCGGCGATTACCGTGAGCCACGTATTATG

CGTTTCGGTTTCACTCCTCTGTATACTACTTTTA

CGGAAGTTTGGGATGCAGTACAAATCCTGGGCG

AAATCCTGGATCGTAAGACTTGGGCGCAGGCTC

AGTTTCAGGTGCGCCACTCTGTTACTTAA [0146] These strains were validated in the checkerboard assay described in

EXAMPLE 15 to have similar ALE parameters to their plasmid-based Ptet counterpart. Lower limit of kynurenine (KYN) and ToxTrp concentration for use in the ALE experiment were established using the checkerboard assay described above herein, and lower limit concentrations corresponded to those observed for the strains expressing tet inducible KYNase from a medium copy plasmid.

[0147] Mutants derived from parental strains SYN2027 and SYN2028 were evolved by passaging in lowering concentrations of KYN and three different ToxTrp concentrations as follows.

[0148] The ALE parental strains were cultured on plates with M9 minimal media supplemented with glucose and L-kynurenine (M9+KYN). A single colony from each parent was selected, resuspended in 20 uL of sterile phosphate-buffered saline solution. This colony was then used to inoculate two cultures of M9+KYN, grown into late-logarithmic phase and the optical density was determined at 600 nm. These cultures were then diluted to 10 in 3 columns of a 96-well deep-well plate with 1 mL of M9+KYNU. Each one of the three rows had different ToxTrp concentrations

(increasing 2-fold), while each column had decreasing concentrations of KYN (by 2- fold). Every 12 hours, the plate was diluted back using 30 uL from the well in which the culture had grown to an OD600 of roughly 0.1. This process was repeated for five days, and then the ToxTrp concentrations were doubled to maintain selection pressure. After two weeks' time, no growth rate increases were detected and the culture was plated onto M9+KYN. All culturing was done shaking at 350 RPM at 37°C. Individual colonies were selected and screened in M9+KYN+ToxTrp media to confirm increased growth rate phenotype.

[0149] Two replicates for each parental strain (SYN20207-R1, SYN2027-R2,

SYN2028-R1, and SYN2028-R2) were selected and assayed for kynurenine production.

[0150] Briefly, overnight cultures were diluted 1: 100 in 400 ml LB and let grow for 4 hours. Next, 2ml of the culture was spun down and resuspended in 2ml M9 buffer. The OD600 of the culture was measured (1/100 dilution in PBS). The necessary amount of cell culture for a 3ml assay targeting starting cell count of ~OD 0.8 (~ 1E8) was spun down. The cell pellet was resuspended in M9+0.5% glucose + 75uM KYN in the assay volume (3ml) in a culture tube. 220ul was removed in triplicate at each time point (t=0, 2, and 3 hours) into conical shaped 96WP, and 4ul were removed for cfu measurement at each time point. At each time point, the sample was spun down in the conical 96WP for 5 minutes at 3000g, and 200 ul were transferred from each well into a clear, flat-bottomed, 96WP. A kynurenine standard curve and blank sample was prepared in the same plate. Next, 40ul of 30% Tri-Chloric Acid (v/v) was added to each well and mixed by pipetting up and down. The plat was sealed with aluminum foil and incubated at 60 C for 15 minutes. The plate was the spun down at 11500rpm, at 4 C, for 15 minutes, and 125ul from each well were aliquoted and mixed with 125 ul of 2% Ehrlich's reagent in glacial acetic acid in another 96WP. Samples were mixed pipetting up and down and the absorbance was measured at OD480. Growth rates are shown for parental strains SYN2027 and SYN2028 and the corresponding evolved strains in FIG. 13.

Example 17. Kynurenine consuming strains decrease tumoral kynurenine levels in the CT26 murine tumor model

[0151] The ability of genetically engineered bacteria comprising kynureninase from Pseudomonas fluorescens to consume kynurenine in vivo in the tumor

environment was assessed. SYN1704, an E. coli Nissle strain comprising a deletion in Trp:E and a medium copy plasmid expressing kynureninase from Pseudomonas fluorescens under control of a constitutive promoter (Nissle delta TrpE::CmR + Pconstitutive-Pseudomonas KYNU KanR) was used for in a first study (Study 1).

[0152] In a second study (Study 2) the activity of SYN2028, an E. coli Nissle strain comprising a deletion in Trp:E and an integrated construct expressing

kynureninase from Pseudomonas fluorescens under the control of a constitutive promoter (Nissle HA3/4::PSynJ23119-pKYNase KanR TrpE::CmR) was assessed.

[0153] In both studies, CT26 cells obtained from ATCC were cultured according to guidlelines provided. Approximately ~le6cells/mouse in PBS were implanted subcutaneously into the right flank of each animal (BalbC/J (female, 8 weeks)), and tumor growth was monitored for approximately 10 days. When the tumors reached about ~100-150mm3, animals were randomized into groups for dosing.

[0154] For intratumoral injection, bacteria were grown in LB broth until reaching an absorbance at 600 nm (A600 nm) of 0.4 (corresponding to 2 X 108 colony- forming units (CFU)/mL) and washed twice in PBS. The suspension was diluted in PBS or saline so that 100 microL can be injected at the appropriate doses intratumorally into tumor-bearing mice.

Study 1:

[0155] Approximately 10 days after CT 26 implanation, bacteria were suspended in 0.1 ml of PBS and mice were injected (5e6 cells/mouse) with 100 ul intratumorally as follows: Group 1-Vehicle Control (n=8), Group 2-SYN94 (n=8), and Group 3-SYN1704 (n=8). From Day 2 until study end, animals were dosed

intratumorally biweekly with lOOul of vehicle control or bacteria at 5e6 cells/mouse. Animals were weighed and the tumor volume measured twice weekly. Animals were euthanized when the tumors reached ~2000mm3 and kynurenine concentrations were measured by LC/MS as described herein. Results are shown in FIG. 14A. A significant reduction in intra tumor concentration was observed for the kynurenine consuming strain SYN1704 and for wild type E. coli Nissle. Intratumoral kynurenine levels were reduced in SYN1704, as compared to wild type Nissle, although the difference did not reach significance due to one outlier.

Study 2:

[0156] Approximately 10 days after CT 26 implanation, bacteria were suspended in 0.1 ml of saline and mice were injected (le8 cells/mouse) with the bacterial suspension intratumorally as follows: Group 1-Vehicle Control (n=10), Group 2-SYN94 (n=10), Group 3-SYN2028 (n=10). Group 5 (n=10) received INCB024360 (IDO inhibitor) via oral gavage as a control twice daily. From Day 2 until study end, animals were dosed intratumorally biweekly with lOOul of vehicle control or bacteria at le8 cells/mouse. Animals were weighed and the tumor volume measured twice weekly. Group 5 received INCB024360 via oral gavage as a control twice daily until study end. Animals were euthanized when the tumors reached ~2000mm3. Tumor fragments were placed in pre-weighed bead-buster tubes and store don ice for analysis. Kynurenine concentrations were measured by LC/MS as described herein. Results are shown in FIG. 14B. A significant reduction in intra tumor concentration was observed for the kynurenine consuming strain SYN2028as compared to wild type Nissle or wild type control. Intratumoral kynurenine levels seen in SYN2028 were similar to those observed for the IDO inhibitor INCB024360. Example 18. Administration of C. novyi expressing human anti-PD-1 antibody, IL- 15, and kynureninase (depleting kynurenine) in humanized mice as a model for colon cancer

[0157] Humanized CD34+ mice (NOD scid gamma mice engrafted with

CD34+ cells through injection of CD34+ hematopoietic stem cells) are purchased from Jackson Labs (Pearson et ah, 2008), bred, and maintained under specific pathogen- free conditions.

[0158] HT-29 cell line is obtained from ATCC and cultured according to the guidelines provided. Approximately 3 X 10⁶ tumor cells are implanted on the flank of Humanized CD34+ mice. Animals are randomized based on tumor size and treatment is initiated when tumors reach 100 mm .

[0159] For i.v. injection and intratumoral injection, bacteria are grown in LB broth until reaching an absorbance at 600 nm (A600 nm) of 0.4 (corresponding to 2 X 10 colony-forming units (CFU)/mL) and washed twice in PBS. The suspension is then diluted to so that 100 microL can be injected at the appropriate doses into the lateral tail vein or intratumorally into tumor-bearing mice. Bacterial spores are suspended in 0.1 ml of PBS for injection. Vehicle control mice are injected with 0.1 mL PBS via tail vein.

[0160] For intratumoral administration, mice are administered either C. novyi expressing human anti-PD-1 antibody, IL-15, and kynureninase, or the control bacterial spores (wild-type C. novyi). A control group is injected with the same volume of PBS. Intratumoral injections are performed 2 to 3 times weekly with various doses of bacteria expressing a anti- human PD-1 antibody or control bacteria, including the optimum dose determined as described above.

[0161] For intravenous administration, mice are intravenously administered either C. novyi expressing human anti-PD-1 antibody, IL-15, and kynureninase, or the control bacteria (wild-type C. novyi) through tail vein injection at doses ranging from 1

X 10 5^J to 1 X 108°. A control group is injected with the same volume of PBS.

[0162] For oral administration, mice are gavaged with 5 X 10⁹ CFU bacteria, either C. novyi expressing human anti-PD-1 antibody, IL-15, and kynureninase, or the control bacteria (wild-type C. novyi), as described in Danino et ah, 2015. [0163] Tumors are measured two to three times per week until study termination. Tumor Volume is calculated (length 9 (width 9 width) 9 0.5) = volume in mm³.

Example 19. Generation of Indole Propionic Acid Strain and in vitro indole production

[0164] To facilitate inducible production of indole propionic acid (IPA) in

Escherichia coli Nissle, 6 genes allowing the production of indole propionic acid from tryptophan, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322 under a tet inducible promoter. In other embodiments, the IPA synthesis cassette is put under the control of an FNR, RNS or ROS promoter, e.g., described herein, or other promoter induced by conditions in the healthy or diseased gut, e.g., inflammatory conditions. For efficient translation of IPA synthesis genes, each synthetic gene in the cassette is separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

[0165] The IPA synthesis cassette comprises TrpDH (tryptophan

dehydrogenase from Nostoc punctiforme NIES-2108), FldHl/FldH2 (indole-3-lactate dehydrogenase from Clostridium sporogenes), FldA (indole-3-propionyl-CoA:indole-3- lactate CoA transferase from Clostridium sporogenes), FldBC (indole-3-lactate dehydratase from Clostridium sporogenes), FldD (indole-3-acrylyl-CoA reductase from Clostridium sporogenes), and Acul (acrylyl-CoA reductase from Rhodobacter sphaeroides).

[0166] The tet inducible IPA construct described above is transformed into

E.coli Nissle as described herein and production of IPA is assessed. In certain embodiments, E. coli Nissle strains containing the IPA synthesis cassette described further comprise a tryptophan synthesis cassette. In certain embodiments, the strains comprise a feedback resistant version of AroG and TrpE to achieve greater Trp production. In certain embodiments, additionally, the tnaA gene (tryptophanase converting Trp into indole) is deleted.

All incubations are performed at 37°C. LB-grown overnight cultures of E. coli Nissle transformed with the IPA biosynthesis construct alone or in combination with a tryptophan biosynthis construct and feedback resistant AroG and TrpE are subcultured 1: 100 into lOmL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2h, at which time anhydrous tetracycline (ATC) is added to cultures at a concentration of lOOng/mL to induce expression of the IPA biosynthesis and tryptophan biosynthesis constructs. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points ( e.g., 0 up to 24 hours) by LC-MS to assess levels of IPA.

[0167] Production of IPA is also assessed in E. coli Nissle strains containing the IPA and tryptophan cassettes both driven by an RNS promoter e.g., a nsrR-norB- IPA biosynthesis construct) in order to assess nitrogen dependent induction of IPA production. Overnight bacterial cultures are diluted 1: 100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine- nitric oxide adduct) wis added to cultures at a final concentration of 0.3mM to induce expression from plasmid. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (0 up to 24 hours, as shown in Fig. 33) to assess IPA levels.

[0168] In alternate embodiments, production of IPA is also assessed in E. coli

Nissle strains containing the IPA and tryptophan cassettes both driven by the low oxygen inducible FNR promoter, e.g., FNRS, or the reactive oxygene regulated OxyS promoter.

Example 20. Synthesis of Constructs for Tryptophan Biosynthesis and Indole Metabolite Synthesis.

[0169] Various constructs are synthesized, and cloned into vector pBR322 for transformation of E. coli. In some embodiments, the constructs encoding the effector molecules are integrated into the genome.

Table 46. Exemplary Sequences and Construct Sequences ofr Tryptophan and

Indole Metabolite Synthesis Description Sequence

fbrAroG (RBS and Ctctagaaataattttgtttaactttaagaaggagatatacat

leader region atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctgga underlined) aaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgcggctaaag

SEQ ID NO: 166 agtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctggaaatcgtgatgcgcgtct attttgaaaagccgcgtactacggtgggctggaaagggctgattaacgatccgcatatggataacagctt ccagatcaacgacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagc ggcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggc gcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgcactgcttcct gtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacggcgattgccatatcattctg cgcggcggtaaagagcctaactacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaa gcaggcctgccagcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcag atggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgtgatggtgg aaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagca tcaccgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgcagtaaaagc gcgtcgcgggtaa

fbr-AroG-serA Ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacgatttacgcatc (RBS and leader aaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccgctactgaaaatgccgcga region underlined; atacggtcgcccatgcccgaaaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtgg SerA starts after tgattggcccatgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgt second RBS) gaagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggc tggaaagggctgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc

SEQ ID NO: 168 gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatgatcaccct acaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccaccgaatcgcaggtgcacc gcgaactggcgtctggtctttcttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggct atcgatgccattaatgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcg attgtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaactacagc gcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatc gatttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttgccagca gattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctggtggaaggcaatcaga gcctcgagagcggggaaccgctggcctacggtaagagcatcaccgatgcctgcattggctgggatga taccgatgctctgttacgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACT taagaaggagatatacatatggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaa ggcgtgcaccaaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaagg cgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctgcgatcccgtac ccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctattggctgtttctgtatcggaaca aatcaggttgatctggatgcggcggcaaagcgcgggatcccggtatttaacgcaccgttctcaaatacg cgctctgttgcggagctggtgattggcgaactgctgctgctattgcgcggcgtgccagaagccaatgct aaagcgcatcgtggcgtgtggaacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgg gtatcatcggctacggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttactt ttatgatattgaaaacaaactgccgctgggcaacgccactcaggtacagcatctttctgacctgctgaata tgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaatatgatgggcgcgaaagag atttcgctaatgaagcccggctcgctgctgattaatgcttcgcgcggtactgtggtggatattccagcgct gtgtgacgcgctggcgagcaaacatctggcgggggcggcaatcgacgtattcccgacggaaccggc gaccaatagcgatccatttacctctccgctgtgtgaattcgacaatgtccttctgacgccacacattggcg gttcgactcaggaagcgcaggagaatatcggcttggaagttgcgggtaaattgatcaagtattctgacaa tggctcaacgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgtcgtctgat gcacatccacgaaaaccgtccgggcgtgctaactgcgctcaacaaaatttttgccgagcagggcgtca acatcgccgcgcaatatctacaaacttccgcccagatgggttatgtagttattgatattgaagccgacgaa gacgttgccgaaaaagcgctgcaggcaatgaaagctattccgggtaccattcgcgcccgtctgctgtac taa

fbrAroG-Tdc (tdc ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacgatttacgcatc from C. roseus); aaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccgctactgaaaatgccgcga RBS and leader atacggtcgcccatgcccgaaaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtgg region underlined tgattggcccatgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgt gaagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggc

SEQ ID NO: 170 tggaaagggctgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatgatcaccct acaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccaccgaatcgcaggtgcacc gcgaactggcgtctggtctttcttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggct atcgatgccattaatgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcg attgtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaactacagc gcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatc gatttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttgccagca gattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctggtggaaggcaatcaga gcctcgagagcggggaaccgctggcctacggtaagagcatcaccgatgcctgcattggctgggatga taccgatgctctgttacgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaag gagatatacatATGGGTTCTATTGACTCGACGAATGTGGCCATGTCT

AATTCTCCTGTTGGCGAGTTTAAGCCCCTTGAAGCAGAAGAGT

TCCGTAAACAGGCACACCGCATGGTGGATTTTATTGCGGATTA

TTACAAGAACGTAGAAACATACCCGGTCCTTTCCGAGGTTGAA

CCCGGCTATCTGCGCAAACGTATTCCCGAAACCGCACCATACC

TGCCGGAGCCACTTGATGATATTATGAAGGATATTCAAAAGG

ACATTATCCCCGGAATGACGAACTGGATGTCCCCGAACTTTTA

CGCCTTCTTCCCGGCCACAGTTAGCTCAGCAGCTTTCTTGGGG

GAAATGCTTTCAACGGCCCTTAACAGCGTAGGATTTACCTGGG

TCAGTTCCCCGGCAGCGACTGAATTAGAGATGATCGTTATGGA

TTGGCTTGCGCAAATTTTGAAACTTCCAAAAAGCTTTATGTTCT

CCGGAACCGGGGGTGGTGTCATCCAAAACACTACGTCAGAGT

CGATCTTGTGCACTATTATCGCGGCCCGTGAACGCGCCTTGGA

AAAATTGGGCCCTGATTCAATTGGTAAGCTTGTCTGCTATGGG

TCCGATCAAACGCACACAATGTTTCCGAAAACCTGTAAGTTAG

CAGGAATTTATCCGAATAATATCCGCCTTATCCCTACCACGGT

AGAAACCGACTTTGGCATCTCACCGCAGGTACTTCGCAAGATG

GTCGAAGACGACGTCGCTGCGGGGTACGTTCCCTTATTTTTGT

GTGCCACCTTGGGAACGACATCAACTACGGCAACAGATCCTGT

AGATTCGCTGTCCGAAATCGCAAACGAGTTTGGTATCTGGATT

CATGTCGACGCCGCATATGCTGGATCGGCTTGCATCTGCCCAG

AATTTCGTCACTACCTTGATGGCATCGAACGTGTGGATTCCTT

ATCGCTGTCTCCCCACAAATGGCTTTTAGCATATCTGGATTGC

ACGTGCTTGTGGGTAAAACAACCTCACCTGCTGCTTCGCGCTT

TAACGACTAATCCCGAATACTTGAAGAATAAACAGAGTGATTT

AGATAAGGTCGTGGATTTTAAGAACTGGCAGATCGCAACAGG

ACGTAAGTTCCGCTCTTTAAAACTTTGGTTAATTCTGCGTTCCT

ACGGGGTAGTTAACCTGCAAAGTCATATCCGTAGTGATGTAGC

GATGGGGAAGATGTTTGAGGAATGGGTCCGTTCCGATAGCCG CTTTGAAATCGTCGTGCCACGTAATTTTTCGCTTGTATGCTTTC

GCTTGAAACCGGATGTATCTAGTTTACATGTCGAGGAGGTCAA

CAAGAAGTTGTTGGATATGCTTAACTCCACCGGTCGCGTATAT

ATGACGCATACAATTGTTGGCGGAATCTATATGTTACGTTTGG

CTGTAGGTAGCAGCTTGACAGAGGAACATCACGTGCGCCGCG

TTTGGGACTTGATCCAGAAGCTTACGGACGACCTGCTTAAAGA

GGCGTGA

fbrAroG-Tdc (tdc ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacgatttacgcatc from Clostridium aaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccgctactgaaaatgccgcga sporo genes); RBS atacggtcgcccatgcccgaaaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtgg and leader region tgattggcccatgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgt underlined gaagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggc tggaaagggctgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc

SEQ ID NO: 172 gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatgatcaccct acaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccaccgaatcgcaggtgcacc gcgaactggcgtctggtctttcttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggct atcgatgccattaatgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcg attgtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaactacagc gcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatc gatttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttgccagca gattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctggtggaaggcaatcaga gcctcgagagcggggaaccgctggcctacggtaagagcatcaccgatgcctgcattggctgggatga taccgatgctctgttacgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaag gagatatacatATGAAATTTTGGCGCAAGTATACGCAACAGGAGATG

GATGAGAAAATCACAGAATCGCTTGAGAAGACATTAAATTAC

GATAACACGAAAACCATCGGCATCCCAGGTACTAAGCTGGAT

GATACTGTATTTTATGACGATCACTCCTTCGTTAAGCACTCTCC

CTATTTACGTACGTTCATCCAAAACCCTAATCACATTGGTTGTC

ACACGTACGATAAAGCAGACATCTTGTTTGGCGGCACGTTTGA

CATCGAACGCGAACTGATTCAGCTTTTGGCCATCGATGTCTTA

AACGGAAATGATGAGGAATTCGATGGATATGTGACACAGGGG

GGAACCGAGGCGAATATTCAGGCAATGTGGGTTTATCGTAACT

ATTTCAAAAAAGAACGTAAAGCAAAACATGAGGAAATCGCAA

TCATCACGAGCGCGGATACCCATTACAGTGCATATAAGGGGA

GCGACTTGCTGAACATTGATATTATCAAGGTCCCAGTAGACTT

CTATTCGCGTAAGATCCAGGAGAACACGTTAGACTCGATTGTC

AAGGAGGCGAAGGAAATTGGAAAGAAGTACTTCATTGTCATC

TCAAACATGGGTACGACTATGTTTGGCAGTGTAGACGACCCTG

ATCTTTATGCTAACATTTTTGATAAGTATAACTTAGAATACAA

AATCCACGTCGATGGAGCTTTTGGGGGTTTCATTTATCCTATC

GATAATAAGGAGTGCAAAACAGATTTCTCGAACAAGAACGTC

TCATCCATCACGCTTGACGGTCACAAAATGCTTCAAGCCCCCT

ATGGGACTGGTATCTTCGTGTCACGTAAGAACTTGATCCATAA

CACCCTGACAAAGGAAGCAACGTATATTGAAAACCTGGACGT

TACCCTGAGTGGGTCCCGCTCCGGATCCAACGCCGTTGCGATC

TGGATGGTTTTAGCCTCTTATGGCCCCTACGGGTGGATGGAGA

AGATTAACAAGTTGCGCAATCGCACTAAGTGGCTTTGCAAGCA

GCTTAACGACATGCGCATCAAATACTATAAGGAGGATAGCAT

GAATATCGTCACGATTGAAGAGCAATACGTAAATAAAGAGAT TGCAGAGAAATACTTCCTTGTGCCTGAAGTACACAATCCTACC AACAATTGGTACAAGATTGTAGTCATGGAACATGTTGAACTTG ACATCTTGAACTCCCTTGTTTATGATTTACGTAAATTCAACAA GGAGCACCTGAAGGCAATGTGA

fbrAroG-trpDH- Ctctagaaataattttgtttaactttaagaaggagatatacat

ipdC-iadl (RBS atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctgga and leader region aaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct underlined) gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgcggctaaag agtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctggaaatcgtgatgcgcgtct

SEQ ID NO: 174 attttgaaaagccgcgtactacggtgggctggaaagggctgattaacgatccgcatatggataacagctt ccagatcaacgacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagc ggcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggc gcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgcactgcttcct gtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacggcgattgccatatcattctg cgcggcggtaaagagcctaactacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaa gcaggcctgccagcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcag atggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgtgatggtgg aaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagca tcaccgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgcagtaaaagc gcgtcgcgggtaaTACTtaagaaggagatatacatATGCTGTTATTCGAGACTGT

GCGTGAAATGGGTCATGAGCAAGTCCTTTTCTGTCATAGCAAG

AATCCCGAGATCAAGGCAATTATCGCAATCCACGATACCACCT

TAGGACCGGCTATGGGCGCAACTCGTATCTTACCTTATATTAA

TGAGGAGGCTGCCCTGAAAGATGCATTACGTCTGTCCCGCGGA

ATGACTTACAAAGCAGCCTGCGCCAATATTCCCGCCGGGGGC

GGCAAAGCCGTCATCATCGCTAACCCCGAAAACAAGACCGAT

GACCTGTTACGCGCATACGGCCGTTTCGTGGACAGCTTGAACG

GCCGTTTCATCACCGGGCAGGACGTTAACATTACGCCCGACGA

CGTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAGGCGTC

TCAGAAAAGTCGGGAGGGCCGGCACCTATCACCTCTCTGGGA

GTATTTTTAGGCATCAAAGCCGCTGTAGAGTCGCGTTGGCAGT

CTAAACGCCTGGATGGCATGAAAGTGGCGGTGCAAGGACTTG

GGAACGTAGGAAAAAATCTTTGTCGCCATCTGCATGAACACG

ATGTACAACTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGA

GGAAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACCGACT

GAAATCTATTCTTTAGATGTTGATATTTTTGCACCGTGTGCACT

TGGGGGTATTTTGAATAGCCATACCATCCCGTTCTTACAAGCC

TCAATCATCGCAGGAGCAGCGAATAACCAGCTGGAGAACGAG

CAACTTCATTCGCAGATGCTTGCGAAAAAGGGTATTCTTTACT

CACCAGACTACGTTATCAATGCAGGAGGACTTATCAATGTTTA

TAACGAAATGATCGGATATGACGAGGAAAAAGCATTCAAACA

AGTTCATAACATCTACGATACGTTATTAGCGATTTTCGAAATT

GCAAAAGAACAAGGTGTAACCACCAACGACGCGGCCCGTCGT

TTAGCAGAGGATCGTATCAACAACTCCAAACGCTCAAAGAGT

AAAGCGATTGCGGCGTGAAATGtaagaaggagatatacatATGCGTACA

CCCTACTGTGTCGCCGATTATCTTTTAGATCGTCTGACGGACTG

CGGGGCCGATCACCTGTTTGGCGTACCGGGCGATTACAACTTG

CAGTTTCTGGACCACGTCATTGACTCACCAGATATCTGCTGGG TAGGGTGTGCGAACGAGCTTAACGCGAGCTACGCTGCTGACG

GATATGCGCGTTGTAAAGGCTTTGCTGCACTTCTTACTACCTTC

GGGGTCGGTGAGTTATCGGCGATGAACGGTATCGCAGGCTCG

TACGCTGAGCACGTCCCGGTATTACACATTGTGGGAGCTCCGG

GTACCGCAGCTCAACAGCGCGGAGAACTGTTACACCACACGC

TGGGCGACGGAGAATTCCGCCACTTTTACCATATGTCCGAGCC

AATTACTGTAGCCCAGGCTGTACTTACAGAGCAAAATGCCTGT

TACGAGATCGACCGTGTTTTGACCACGATGCTTCGCGAGCGCC

GTCCCGGGTATTTGATGCTGCCAGCCGATGTTGCCAAAAAAGC

TGCGACGCCCCCAGTGAATGCCCTGACGCATAAACAAGCTCAT

GCCGATTCCGCCTGTTTAAAGGCTTTTCGCGATGCAGCTGAAA

ATAAATTAGCCATGTCGAAACGCACCGCCTTGTTGGCGGACTT

TCTGGTCCTGCGCCATGGCCTTAAACACGCCCTTCAGAAATGG

GTCAAAGAAGTCCCGATGGCCCACGCTACGATGCTTATGGGTA

AGGGGATTTTTGATGAACGTCAAGCGGGATTTTATGGAACTTA

TTCCGGTTCGGCGAGTACGGGGGCGGTAAAGGAAGCGATTGA

GGGAGCCGACACAGTTCTTTGCGTGGGGACACGTTTCACCGAT

ACACTGACCGCTGGATTCACACACCAACTTACTCCGGCACAAA

CGATTGAGGTGCAACCCCATGCGGCTCGCGTGGGGGATGTAT

GGTTTACGGGCATTCCAATGAATCAAGCCATTGAGACTCTTGT

CGAGCTGTGCAAACAGCACGTCCACGCAGGACTGATGAGTTC

GAGCTCTGGGGCGATTCCTTTTCCACAACCAGATGGTAGTTTA

ACTCAAGAAAACTTCTGGCGCACATTGCAAACCTTTATCCGCC

CAGGTGATATCATCTTAGCAGACCAGGGTACTTCAGCCTTTGG

AGCAATTGACCTGCGCTTACCAGCAGACGTGAACTTTATTGTG

CAGCCGCTGTGGGGGTCTATTGGTTATACTTTAGCTGCGGCCT

TCGGAGCGCAGACAGCGTGTCCAAACCGTCGTGTGATCGTATT

GACAGGAGATGGAGCAGCGCAGTTGACCATTCAGGAGTTAGG

CTCGATGTTACGCGATAAGCAGCACCCCATTATCCTGGTCCTG

AACAATGAGGGGTATACAGTTGAACGCGCCATTCATGGTGCG

GAACAACGCTACAATGACATCGCTTTATGGAATTGGACGCAC

ATCCCCCAAGCCTTATCGTTAGATCCCCAATCGGAATGTTGGC

GTGTGTCTGAAGCAGAGCAACTGGCTGATGTTCTGGAAAAAG

TTGCTCATCATGAACGCCTGTCGTTGATCGAGGTAATGTTGCC

CAAGGCCGATATCCCTCCGTTACTGGGAGCCTTGACCAAGGCT

TTAGAAGCCTGCAACAACGCTTAAAGGTtaagaaggagatatacatATG

CCCACCTTGAACTTGGACTTACCCAACGGTATTAAGAGCACGA

TTCAGGCAGACCTTTTCATCAATAATAAGTTTGTGCCGGCGCT

TGATGGGAAAACGTTCGCAACTATTAATCCGTCTACGGGGAA

AGAGATCGGACAGGTGGCAGAGGCTTCGGCGAAGGATGTGGA

TCTTGCAGTTAAGGCCGCGCGTGAGGCGTTTGAAACTACTTGG

GGGGAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTGATTA

AGCTTGCTGAGTTGGTGGAAGCGAATATTGATGAGTTAGCGGC

AATTGAATCACTGGACAATGGGAAAGCGTTCTCTATTGCTAAG

TCATTCGACGTAGCTGCTGTGGCCGCAAACTTACGTTACTACG

GCGGTTGGGCTGATAAAAACCACGGTAAAGTCATGGAGGTAG

ACACAAAGCGCCTGAACTATACCCGCCACGAGCCGATCGGGG

TTTGCGGACAAATCATTCCGTGGAATTTCCCGCTTTTGATGTTT

GCATGGAAGCTGGGTCCCGCTTTAGCCACAGGGAACACAATT GTGTTAAAGACTGCCGAGCAGACTCCCTTAAGTGCTATCAAGA

TGTGTGAATTAATCGTAGAAGCCGGCTTTCCGCCCGGAGTAGT

TAATGTGATCTCGGGATTCGGACCGGTGGCGGGGGCCGCGAT

CTCGCAACACATGGACATCGATAAGATTGCCTTTACAGGATCG

ACATTGGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGACTA

ACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAAATCCCCGA

ATATCATTTTCAAAGATGCCGACCTTGACCAAGCTGTTCGCTG

GAGCGCCTTCGGTATCATGTTTAACCACGGACAATGCTGCTGC

GCTGGATCGCGCGTATATGTGGAAGAATCCATCTATGACGCCT

TCATGGAAAAAATGACTGCGCATTGTAAGGCGCTTCAAGTTGG

AGATCCTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTCG

CAGTTACAATACGACCGTATCATGGAATACATCGAATCAGGG

AAAAAAGATGCAAATCTTGCTTTAGGCGGCGTTCGCAAAGGG

AATGAGGGGTATTTCATTGAGCCAACTATTTTTACAGACGTGC

CGCACGACGCGAAGATTGCCAAAGAGGAGATCTTCGGTCCAG

TGGTTGTTGTGTCGAAATTTAAGGACGAAAAAGATCTGATCCG

TATCGCAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTTT

CCCGCGACATCAGCCGCGCGATCGAGACAGCACACAAACTGA

AAGCAGGCACGGTCTGGGTCAACTGCTATAATCAGCTTATTCC

GCAGGTGCCATTCGGAGGGTATAAGGCTTCCGGTATCGGCCGT

GAGTTGGGGGAATATGCCTTGTCTAATTACACAAATATCAAGG

CCGTCCACGTTAACCTTTCTCAACCGGCGCCCATTTGA

TrpEDCBA (RBS Ctctagaaataattttgtttaactttaagaaggagatatacat

and leader region atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgc underlined) gctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaatccgcagatatcgacagcaaa gatgatttaaaaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaa

SEQ ID NO: 178 tccaggcgctttccggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtgg aaaatgaacaatcaccaaactgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacg cccgcttatgctccctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaaga acgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttaccgcaact gtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgaccatcagaa aaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgctcg cctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtttccgtgccgcata tgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcg cgccggagaaattttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcc tattacgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccctgtttg gcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgcc ggaacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgcatcgaac tggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatctcgcccgtaatgacctg gcacgcatttgcacacccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtga tgcacctagtctcccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcct gtatgaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaa ggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctg cattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtcct tgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgcc accgcgcatcatgcacaggagacgttctaatggctgacattctgctgctcgataatatcgactcttttacgt acaacctggcagatcagttgcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgc agaccttaattgaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggtgtgcc gagcgaagccggttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttgc ctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcttcacggt aaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaaacccgctgccagtggc gcgttatcactcgctggttggcagtaacattccggccggtttaaccatcaacgcccattttaatggcatggt gatggcggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccattcttactaccc agggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacgc tgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgtttt cagcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcatgaaaattc gcggtgaacacccgaacgagatcgccggggcagcaaccgcgctactggaaaacgccgcgccattcc cgcgcccggattatctgtttgccgatatcgtcggtactggcggtgacggcagcaacagcatcaatatttct accgccagtgcgtttgtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtct ccagtaaatccggctcgtcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcg cgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatg cgatgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctgggaccattgattaacccgg cgcatccgccgctggcgctaattggtgtttatagtccggaactggtgctgccgattgccgaaaccttgcg cgtgctggggtatcaacgcgcggcagtggtgcacagcggcgggatggatgaagtttcattacacgcg ccgacaatcgttgccgaactacatgacggcgaaattaagagctatcaattgaccgctgaagattttggcc tgacaccctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacacg cttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgc gcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtggttccg cttacgacagagtcaccgcactggcggcacgagggtaaatgatgcaaaccgttttagcgaaaatcgtc gcagacaaggcgatttgggtagaaacccgcaaagagcagcaaccgctggccagttttcagaatgagg ttcagccgagcacgcgacatttttatgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaa aaagcgtcgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccatttataaac attacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcctccccatcgt cagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctggcgc gctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaatatcgccagcttgca gccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagtaatgaagaggaactggagcgcg ccattgcattgggggcaaaggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaa ccgtacccgcgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaat acttacgctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatggccca tgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtgg gcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgcgacatcaccg cgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccgttgcagtatgttggcgtgttc cgcaatcacgatattgccgatgtggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcat ggtaatgaagatcagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaag gctttaagtgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggt cagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctg gcggggggcttaggcgcagataactgcgtggaagcggcacaaaccggctgcgccgggcttgatttta attctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctcggttttccagacgctgcgc gcatattaaggaaaggaacaatgacaacattacttaacccctattttggtgagtttggcggcatgtacgtg ccacaaatcctgatgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaa tttcaggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgaccaaatgccag aacattacagccgggacgaacaccacgctgtatctgaagcgcgaagatttgctgcacggcggcgcgc ataaaactaaccaggtgctcggtcaggctttactggcgaagcggatgggtaaaactgaaattattgccg aaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcgccctgctcggcctgaaatgccg aatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgcg gaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcgactg gtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacccgaccat tgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagagaaggtcgcc tgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatcaacg aaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcg caccgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacg ggcaaattgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtat ctcaacagcactggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacg ctttgcctgcatgaagggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatg atgcgcgaaaatccggaaaaagagcagctactggtggttaacctttccggtcgcggcgataaagacat cttcaccgttcacgatattttgaaagcacgaggggaaatctgatggaacgctacgaatctctgtttgccca gttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctcggtgatccgggcattgagcagtc gttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagttaggcatccccttctccgac ccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccccgg cgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggccttttgatgtat gccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcgattcgg tgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggtt acacctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcat ctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatc aggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcg agcaacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcg gcgacgcgcagtta

fbrS40FTrpE- ctctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactctcgaact DCBA (leader gctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtggggatcgtccg region and RBS gcaacgctgctgctggaattcgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagac underlined) agtgcgctgcgcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagcc ctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgta

SEQ ID NO: 184 ctgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgct ttccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcggcggcctgtt ctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaaaatagctgccctgatttctg tttttatctcgctgaaacgctgatggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtt tgctccgaatgaagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccg aagccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaa gagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccat ctcgccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgctgaaaaagagtaatcccag cccgtacatgttttttatgcaggataatgatttcaccctgtttggcgcgtcgccggaaagttcgctcaagtat gacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtcgtcgtgccga tggttcgctggacagagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttc tgaacatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagccgcta cgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctcccgcgttgttggtgagct gcgccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagcggtgcac cgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcggcagctacggcggcgc ggtaggttattttaccgcgcatggcgatctcgacacctgcattgtgatccgctcggcgctggtggaaaac ggtatcgccaccgtgcaagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaa actcgtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagacgttctaa tggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatg gtcataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcgacgatgag caatccggtgctgatgctttctcctggccccggtgtgccgagcgaagccggttgtatgccggaactcctc acccgcttgcgtggcaagctgccaattattggcatttgcctcggacatcaggcgattgtcgaagcttacg ggggctatgtcggtcaggcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcag gcgatgtttgccggattaacaaacccgctgccagtggcgcgttatcactcgctggttggcagtaacattc cggccggtttaaccatcaacgcccattttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgt ttgtggattccagttccatccggaatccattcttactacccagggcgctcgcctgctggaacaaacgctg gcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattctggaaaaactgtatcagg cacagacgcttagccaacaagaaagccaccagctgttttcagcggtggtacgtggcgagctgaagcc ggaacaactggcggcggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccgg ggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgt cggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctg cgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctgg cggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctg tttcctctttgcgccgaagtatcacaccggattccgccatgcgatgccggttcgccagcaactgaaaacc cgcactctgttcaacgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgttta tagtccggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtgg tgcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggc gaaattaagagctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagcaattggca ggcggaacaccggaagaaaaccgtgacattttaacacgcttgttacaaggtaaaggcgacgccgccc atgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcctgcatggccatgaagatctgcaagcc aatgcgcaaaccgttcttgaggtactgcgcagtggttccgcttacgacagagtcaccgcactggcggca cgagggtaaatgatgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccg caaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgc acttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtgatcc gtgatgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaatttcagtgctgactg atgagaaatattttcaggggagctttgatttcctccccatcgtcagccaaatcgccccgcagccgattttat gtaaagacttcattatcgatccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaat gctttcagtactggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgt gctgaccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggc atcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaactg gggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggtgcgcgagttaagcca cttcgctaacggctttctgattggttcggcgttgatggcccatgacgatttgaacgccgccgtgcgtcgg gtgttgctgggtgagaataaagtatgtggcctgacacgtgggcaagatgctaaagcagcttatgacgcg ggcgcgatttacggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcagg aagtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcgga caaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaat ctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaactcttcccgcg cgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcgggagcggacaacgtttcgac tggtcactattaaatggtcaatcgcttggcaacgttctgctggcggggggcttaggcgcagataactgcg tggaagcggcacaaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatca aagacgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaatgacaac attacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgcca gctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaa aactatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgc tgtatctgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctt tactggcgaagcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcg tcggcccttgccagcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgc cagtcgcccaacgttttccggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcg accctgaaagatgcctgtaatgaggcgctacgcgactggtccggcagttatgaaaccgcgcactatatg ctgggtaccgcagctggcccgcatccttacccgaccattgtgcgtgagtttcagcggatgattggcgaa gaaacgaaagcgcagattctggaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcgg tggttcgaatgccatcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagc ctggcggccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtgggcatctat ttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaagagtcttactccatttctgccg ggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcactggacgcgctgattacgtgt ctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaagggatcatcccggcgct ggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagcta ctggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgagg ggaaatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgtt cctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccgg tgctgacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattcaaaacg ccacactgcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgctggcactcattcgcc agaagcacccgaccattcccatcggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgag ttttatgccgagtgcgagaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtcc gcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacg atgatttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcgcgagcgggcgtgacc ggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacgctgc gcctccattgcagggatttggtatttccgccccggatcaggtaaaagccgcgattgatgcaggagctgc gggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagccagagaaaatgctg gcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa

fbrAroG-trpDH- ctctagaaataattttgtttaactttaagaaggagatatacatatgaattatcagaacgacgatttacgcatc fldABCDacuffldH aaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccgctactgaaaatgccgcga (leader region and atacggtcgcccatgcccgaaaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtgg RBS underlined) tgattggcccatgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgt gaagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggc

SEQ ID NO: 186 tggaaagggctgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatgatcaccct acaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccaccgaatcgcaggtgcacc gcgaactggcgtctggtctttcttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggct atcgatgccattaatgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcg attgtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaactacagc gcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatc gatttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttgccagca gattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctggtggaaggcaatcaga gcctcgagagcggggaaccgctggcctacggtaagagcatcaccgatgcctgcattggctgggatga taccgatgctctgttacgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACTtaagaag gagatatacatATGCTGTTATTCGAGACTGTGCGTGAAATGGGTCAT

GAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCGAGATCAAGG

CAATTATCGCAATCCACGATACCACCTTAGGACCGGCTATGGG

CGCAACTCGTATCTTACCTTATATTAATGAGGAGGCTGCCCTG

AAAGATGCATTACGTCTGTCCCGCGGAATGACTTACAAAGCA

GCCTGCGCCAATATTCCCGCCGGGGGCGGCAAAGCCGTCATC

ATCGCTAACCCCGAAAACAAGACCGATGACCTGTTACGCGCA

TACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTCATCACCG

GGCAGGACGTTAACATTACGCCCGACGACGTTCGCACTATTTC

GCAGGAGACTAAGTACGTGGTAGGCGTCTCAGAAAAGTCGGG

AGGGCCGGCACCTATCACCTCTCTGGGAGTATTTTTAGGCATC

AAAGCCGCTGTAGAGTCGCGTTGGCAGTCTAAACGCCTGGAT

GGCATGAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAAA

AATCTTTGTCGCCATCTGCATGAACACGATGTACAACTTTTTGT

GTCTGATGTCGATCCAATCAAGGCCGAGGAAGTAAAACGCTT

ATTCGGGGCGACTGTTGTCGAACCGACTGAAATCTATTCTTTA GATGTTGATATTTTTGCACCGTGTGCACTTGGGGGTATTTTGA

ATAGCCATACCATCCCGTTCTTACAAGCCTCAATCATCGCAGG

AGCAGCGAATAACCAGCTGGAGAACGAGCAACTTCATTCGCA

GATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACTACGTT

ATCAATGCAGGAGGACTTATCAATGTTTATAACGAAATGATCG

GATATGACGAGGAAAAAGCATTCAAACAAGTTCATAACATCT

ACGATACGTTATTAGCGATTTTCGAAATTGCAAAAGAACAAG

GTGTAACCACCAACGACGCGGCCCGTCGTTTAGCAGAGGATC

GTATCAACAACTCCAAACGCTCAAAGAGTAAAGCGATTGCGG

CGTGAAATGtaagaaggagatatacatATGGAAAACAACACCAATATGT

TCTCTGGAGTGAAGGTGATCGAACTGGCCAACTTTATCGCTGC

TCCGGCGGCAGGTCGCTTCTTTGCTGATGGGGGAGCAGAAGTA

ATTAAGATCGAATCTCCAGCAGGCGACCCGCTGCGCTACACG

GCCCCATCAGAAGGACGCCCGCTTTCTCAAGAGGAAAACACA

ACGTATGATTTGGAAAACGCGAATAAGAAAGCAATTGTTCTG

AACTTAAAATCGGAAAAAGGAAAGAAAATTCTTCACGAGATG

CTTGCTGAGGCAGACATCTTGTTAACAAATTGGCGCACGAAAG

CGTTAGTCAAACAGGGGTTAGATTACGAAACACTGAAAGAGA

AGTATCCAAAATTGGTATTTGCACAGATTACAGGATACGGGG

AGAAAGGACCCGACAAAGACCTGCCTGGTTTCGACTACACGG

CGTTTTTCGCCCGCGGAGGAGTCTCCGGTACATTATATGAAAA

AGGAACTGTCCCTCCTAATGTGGTACCGGGTCTGGGTGACCAC

CAGGCAGGAATGTTCTTAGCTGCCGGTATGGCTGGTGCGTTGT

ATAAGGCCAAAACCACCGGACAAGGCGACAAAGTCACCGTTA

GTCTGATGCATAGCGCAATGTACGGCCTGGGAATCATGATTCA

GGCAGCCCAGTACAAGGACCATGGGCTGGTGTACCCGATCAA

CCGTAATGAAACGCCTAATCCTTTCATCGTTTCATACAAGTCC

AAAGATGATTACTTTGTCCAAGTTTGCATGCCTCCCTATGATG

TGTTTTATGATCGCTTTATGACGGCCTTAGGACGTGAAGACTT

GGTAGGTGACGAACGCTACAATAAGATCGAGAACTTGAAGGA

TGGTCGCGCAAAAGAAGTCTATTCCATCATCGAACAACAAAT

GGTAACGAAGACGAAGGACGAATGGGACAAGATTTTTCGTGA

TGCAGACATTCCATTCGCTATTGCCCAAACGTGGGAAGATCTT

TTAGAAGACGAGCAGGCATGGGCCAACGACTACCTGTATAAA

ATGAAGTATCCCACAGGCAACGAACGTGCCCTGGTACGTTTAC

CTGTGTTCTTCAAAGAAGCTGGACTTCCTGAATACAACCAGTC

GCCACAGATTGCTGAGAATACCGTGGAAGTGTTAAAGGAGAT

GGGATATACCGAGCAAGAAATTGAGGAGCTTGAGAAAGACAA

AGACATCATGGTACGTAAAGAGAAATGAAGGTtaagaaggagatatac atATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAAGGCTA

AACACTATCTGCGCGAGATCACAGCTAAACACTACAAGGAAG

CGTTAGAGGCTAAAGAGCGTGGGGAGAAAGTGGGTTGGTGTG

CCTCTAACTTCCCCCAAGAGATTGCAACCACGTTGGGTGTAAA

GGTTGTTTATCCCGAAAACCACGCCGCCGCCGTAGCGGCACGT

GGCAATGGGCAAAATATGTGCGAACACGCGGAGGCTATGGGA

TTCAGTAATGATGTGTGTGGATATGCACGTGTAAATTTAGCCG

TAATGGACATCGGCCATAGTGAAGATCAACCTATTCCAATGCC

TGATTTCGTTCTGTGCTGTAATAATATCTGCAATCAGATGATTA

AATGGTATGAACACATTGCAAAAACGTTGGATATTCCTATGAT CCTTATCGATATTCCATATAATACTGAGAACACGGTGTCTCAG

GACCGCATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA

AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACGAGAATA

AATTCGAAGAAGTGATGAAGATTTCGCAAGAATCGGCCAAGC

AATGGTTACGCGCCGCGAGCTACGCGAAATACAAACCATCAC

CGTTTTCGGGCTTTGACCTTTTTAATCACATGGCTGTAGCCGTT

TGTGCTCGCGGCACCCAGGAAGCCGCCGATGCATTCAAAATGT

TAGCAGATGAATATGAAGAGAACGTTAAGACAGGAAAGTCTA

CTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCGAGGGCAT

CGCTTGTTGGCCTTATCTGCGCCACAAGTTGACGAAACTGAGT

GAATATGGAATGAACGTCACAGCTACGGTGTACGCCGAAGCT

TTTGGGGTTATTTACGAAAACATGGATGAACTGATGGCCGCTT

ACAATAAAGTGCCTAACTCAATCTCCTTCGAGAACGCGCTGAA

GATGCGTCTTAATGCCGTTACAAGCACCAATACAGAAGGGGC

TGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTCAGGATTC

TTATACGAACTGGCCCGTCGTTTGGAAAAGGAGACGGGGATC

CCTGTTGTTTCGTTCGACGGAGATCAAGCGGATCCCCGTAACT

TCTCCGAGGCTCAATATGACACTCGCATCCAAGGTTTAAATGA

GGTGATGGTCGCGAAAAAAGAAGCAGAGTGAGCTTtaagaaggaga tatacatATGTCGAATAGTGACAAGTTTTTTAACGACTTCAAGGAC

ATTGTGGAAAACCCAAAGAAGTATATCATGAAGCATATGGAA

CAAACGGGACAAAAAGCCATCGGTTGCATGCCTTTATACACCC

CAGAAGAGCTTGTCTTAGCGGCGGGTATGTTTCCTGTTGGAGT

ATGGGGCTCGAATACTGAGTTGTCAAAAGCCAAGACCTACTTT

CCGGCTTTTATCTGTTCTATCTTGCAAACTACTTTAGAAAACGC

ATTGAATGGGGAGTATGACATGCTGTCTGGTATGATGATCACA

AACTATTGCGATTCGCTGAAATGTATGGGACAAAACTTCAAAC

TTACAGTGGAAAATATCGAATTCATCCCGGTTACGGTTCCACA

AAACCGCAAGATGGAGGCGGGTAAAGAATTTCTGAAATCCCA

GTATAAAATGAATATCGAACAACTGGAAAAAATCTCAGGGAA

TAAGATCACTGACGAGAGCTTGGAGAAGGCTATTGAAATTTA

CGATGAGCACCGTAAAGTCATGAACGATTTCTCTATGCTTGCG

TCCAAGTACCCTGGTATCATTACGCCAACGAAACGTAACTACG

TGATGAAGTCAGCGTATTATATGGACAAGAAAGAACATACAG

AGAAGGTACGTCAGTTGATGGATGAAATCAAGGCCATTGAGC

CTAAACCATTCGAAGGAAAACGCGTGATTACCACTGGGATCA

TTGCAGATTCGGAGGACCTTTTGAAAATCTTGGAGGAGAATAA

CATTGCTATCGTGGGAGATGATATTGCACACGAGTCTCGCCAA

TACCGCACTTTGACCCCGGAGGCCAACACACCTATGGACCGTC

TTGCTGAACAATTTGCGAACCGCGAGTGTTCGACGTTGTATGA

CCCTGAAAAAAAACGTGGACAGTATATTGTCGAGATGGCAAA

AGAGCGTAAGGCCGACGGAATCATCTTCTTCATGACAAAATTC

TGCGATCCCGAAGAATACGATTACCCTCAGATGAAAAAAGAC

TTCGAAGAAGCCGGTATTCCCCACGTTCTGATTGAGACAGACA

TGCAAATGAAGAACTACGAACAAGCTCGCACCGCTATTCAAG

CATTTTCAGAAACCCTTTGACGCTtaagaaggagatatacatATGCGTGC

TGTCTTAATCGAGAAGTCAGATGACACCCAGAGTGTTTCAGTT

ACGGAGTTGGCTGAAGACCAATTACCCGAAGGTGACGTCCTT

GTGGATGTCGCGTACAGCACATTGAATTACAAGGATGCTCTTG CGATTACTGGAAAAGCACCCGTTGTACGCCGTTTTCCTATGGT

CCCCGGAATTGACTTTACTGGGACTGTCGCACAGAGTTCCCAT

GCTGATTTCAAGCCAGGCGACCGCGTAATTCTGAACGGATGG

GGAGTTGGTGAGAAACACTGGGGCGGTCTTGCAGAACGCGCA

CGCGTACGTGGGGACTGGCTTGTCCCGTTGCCAGCCCCCTTAG

ACTTGCGCCAGGCTGCAATGATTGGCACTGCGGGGTACACAG

CTATGCTGTGCGTGCTTGCCCTTGAGCGCCATGGAGTCGTACC

TGGGAACGGCGAGATTGTCGTCTCAGGCGCAGCAGGAGGGGT

AGGTTCTGTAGCAACCACACTGTTAGCAGCCAAAGGCTACGA

AGTGGCCGCCGTGACCGGGCGCGCAAGCGAGGCCGAATATTT

ACGCGGATTAGGCGCCGCGTCGGTCATTGATCGCAATGAATTA

ACGGGGAAGGTGCGTCCATTAGGGCAGGAACGCTGGGCAGGA

GGAATCGATGTAGCAGGATCAACCGTACTTGCTAATATGTTGA

GCATGATGAAATACCGTGGCGTGGTGGCGGCCTGTGGCCTGG

CGGCTGGAATGGACTTGCCCGCGTCTGTCGCCCCTTTTATTCTG

CGTGGTATGACTTTGGCAGGGGTAGATTCAGTCATGTGCCCCA

AAACTGATCGTCTGGCTGCTTGGGCACGCCTGGCATCCGACCT

GGACCCTGCAAAGCTGGAAGAGATGACAACTGAATTACCGTT

CTCTGAGGTGATTGAAACGGCTCCGAAGTTCTTGGATGGAACA

GTGCGTGGGCGTATTGTCATTCCGGTAACACCTTGATACTtaaga aggagatatacatATGAAAATCTTGGCATACTGCGTCCGCCCAGACGA

GGTAGACTCCTTTAAGAAATTTAGTGAAAAGTACGGGCATAC

AGTTGATCTTATTCCAGACTCTTTTGGACCTAATGTCGCTCATT

TGGCGAAGGGTTACGATGGGATTTCTATTCTGGGCAACGACAC

GTGTAACCGTGAGGCACTGGAGAAGATCAAGGATTGCGGGAT

CAAATATCTGGCAACCCGTACAGCCGGAGTGAACAACATTGA

CTTCGATGCAGCAAAGGAGTTCGGTATTAACGTGGCTAATGTT

CCCGCATATTCCCCCAACTCGGTCAGCGAATTTACCATTGGAT

TGGCATTAAGTCTGACGCGTAAGATTCCATTTGCCCTGAAACG

CGTGGAACTGAACAATTTTGCGCTTGGCGGCCTTATTGGTGTG

GAATTGCGTAACTTAACTTTAGGAGTCATCGGTACTGGTCGCA

TCGGATTGAAAGTGATTGAGGGCTTCTCTGGGTTTGGAATGAA

AAAAATGATCGGTTATGACATTTTTGAAAATGAAGAAGCAAA

GAAGTACATCGAATACAAATCATTAGACGAAGTTTTTAAAGA

GGCTGATATTATCACTCTGCATGCGCCTCTGACAGACGACAAC

TATCATATGATTGGTAAAGAATCCATTGCTAAAATGAAGGATG

GGGTATTTATTATCAACGCAGCGCGTGGAGCCTTAATCGATAG

TGAGGCCCTGATTGAAGGGTTAAAATCGGGGAAGATT

fbrAroG-TrpDH- Ctctagaaataattttgtttaactttaagaaggagatatacat

fldABCDH (RBS atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctgga and leader region aaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgcggctaaag

SEQ ID NO: 192 agtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctggaaatcgtgatgcgcgtct attttgaaaagccgcgtactacggtgggctggaaagggctgattaacgatccgcatatggataacagctt ccagatcaacgacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagc ggcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggc gcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgcactgcttcct gtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacggcgattgccatatcattctg cgcggcggtaaagagcctaactacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaa gcaggcctgccagcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcag atggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgtgatggtgg aaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagca tcaccgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgcagtaaaagc gcgtcgcgggtaaTACTtaagaaggagatatacatATGCTGTTATTCGAGACTGT

GCGTGAAATGGGTCATGAGCAAGTCCTTTTCTGTCATAGCAAG

AATCCCGAGATCAAGGCAATTATCGCAATCCACGATACCACCT

TAGGACCGGCTATGGGCGCAACTCGTATCTTACCTTATATTAA

TGAGGAGGCTGCCCTGAAAGATGCATTACGTCTGTCCCGCGGA

ATGACTTACAAAGCAGCCTGCGCCAATATTCCCGCCGGGGGC

GGCAAAGCCGTCATCATCGCTAACCCCGAAAACAAGACCGAT

GACCTGTTACGCGCATACGGCCGTTTCGTGGACAGCTTGAACG

GCCGTTTCATCACCGGGCAGGACGTTAACATTACGCCCGACGA

CGTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAGGCGTC

TCAGAAAAGTCGGGAGGGCCGGCACCTATCACCTCTCTGGGA

GTATTTTTAGGCATCAAAGCCGCTGTAGAGTCGCGTTGGCAGT

CTAAACGCCTGGATGGCATGAAAGTGGCGGTGCAAGGACTTG

GGAACGTAGGAAAAAATCTTTGTCGCCATCTGCATGAACACG

ATGTACAACTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGA

GGAAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACCGACT

GAAATCTATTCTTTAGATGTTGATATTTTTGCACCGTGTGCACT

TGGGGGTATTTTGAATAGCCATACCATCCCGTTCTTACAAGCC

TCAATCATCGCAGGAGCAGCGAATAACCAGCTGGAGAACGAG

CAACTTCATTCGCAGATGCTTGCGAAAAAGGGTATTCTTTACT

CACCAGACTACGTTATCAATGCAGGAGGACTTATCAATGTTTA

TAACGAAATGATCGGATATGACGAGGAAAAAGCATTCAAACA

AGTTCATAACATCTACGATACGTTATTAGCGATTTTCGAAATT

GCAAAAGAACAAGGTGTAACCACCAACGACGCGGCCCGTCGT

TTAGCAGAGGATCGTATCAACAACTCCAAACGCTCAAAGAGT

AAAGCGATTGCGGCGTGAAATGtaagaaggagatatacatATGGAAAAC

AACACCAATATGTTCTCTGGAGTGAAGGTGATCGAACTGGCCA

ACTTTATCGCTGCTCCGGCGGCAGGTCGCTTCTTTGCTGATGG

GGGAGCAGAAGTAATTAAGATCGAATCTCCAGCAGGCGACCC

GCTGCGCTACACGGCCCCATCAGAAGGACGCCCGCTTTCTCAA

GAGGAAAACACAACGTATGATTTGGAAAACGCGAATAAGAAA

GCAATTGTTCTGAACTTAAAATCGGAAAAAGGAAAGAAAATT

CTTCACGAGATGCTTGCTGAGGCAGACATCTTGTTAACAAATT

GGCGCACGAAAGCGTTAGTCAAACAGGGGTTAGATTACGAAA

CACTGAAAGAGAAGTATCCAAAATTGGTATTTGCACAGATTAC

AGGATACGGGGAGAAAGGACCCGACAAAGACCTGCCTGGTTT

CGACTACACGGCGTTTTTCGCCCGCGGAGGAGTCTCCGGTACA

TTATATGAAAAAGGAACTGTCCCTCCTAATGTGGTACCGGGTC

TGGGTGACCACCAGGCAGGAATGTTCTTAGCTGCCGGTATGGC

TGGTGCGTTGTATAAGGCCAAAACCACCGGACAAGGCGACAA

AGTCACCGTTAGTCTGATGCATAGCGCAATGTACGGCCTGGGA

ATCATGATTCAGGCAGCCCAGTACAAGGACCATGGGCTGGTG

TACCCGATCAACCGTAATGAAACGCCTAATCCTTTCATCGTTT

CATACAAGTCCAAAGATGATTACTTTGTCCAAGTTTGCATGCC TCCCTATGATGTGTTTTATGATCGCTTTATGACGGCCTTAGGAC

GTGAAGACTTGGTAGGTGACGAACGCTACAATAAGATCGAGA

ACTTGAAGGATGGTCGCGCAAAAGAAGTCTATTCCATCATCGA

ACAACAAATGGTAACGAAGACGAAGGACGAATGGGACAAGA

TTTTTCGTGATGCAGACATTCCATTCGCTATTGCCCAAACGTG

GGAAGATCTTTTAGAAGACGAGCAGGCATGGGCCAACGACTA

CCTGTATAAAATGAAGTATCCCACAGGCAACGAACGTGCCCT

GGTACGTTTACCTGTGTTCTTCAAAGAAGCTGGACTTCCTGAA

TACAACCAGTCGCCACAGATTGCTGAGAATACCGTGGAAGTG

TTAAAGGAGATGGGATATACCGAGCAAGAAATTGAGGAGCTT

GAGAAAGACAAAGACATCATGGTACGTAAAGAGAAATGAAG

GTtaagaaggagatatacatATGTCAGACCGCAACAAAGAAGTGAAAGA

AAAGAAGGCTAAACACTATCTGCGCGAGATCACAGCTAAACA

CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGAGAAAGT

GGGTTGGTGTGCCTCTAACTTCCCCCAAGAGATTGCAACCACG

TTGGGTGTAAAGGTTGTTTATCCCGAAAACCACGCCGCCGCCG

TAGCGGCACGTGGCAATGGGCAAAATATGTGCGAACACGCGG

AGGCTATGGGATTCAGTAATGATGTGTGTGGATATGCACGTGT

AAATTTAGCCGTAATGGACATCGGCCATAGTGAAGATCAACCT

ATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATATCTGCAA

TCAGATGATTAAATGGTATGAACACATTGCAAAAACGTTGGAT

ATTCCTATGATCCTTATCGATATTCCATATAATACTGAGAACA

CGGTGTCTCAGGACCGCATTAAGTACATCCGCGCCCAGTTCGA

TGACGCTATCAAGCAACTGGAAGAAATCACTGGCAAAAAGTG

GGACGAGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAGA

ATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACGCGAAATA

CAAACCATCACCGTTTTCGGGCTTTGACCTTTTTAATCACATGG

CTGTAGCCGTTTGTGCTCGCGGCACCCAGGAAGCCGCCGATGC

ATTCAAAATGTTAGCAGATGAATATGAAGAGAACGTTAAGAC

AGGAAAGTCTACTTATCGCGGCGAGGAGAAGCAGCGTATCTT

GTTCGAGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTTG

ACGAAACTGAGTGAATATGGAATGAACGTCACAGCTACGGTG

TACGCCGAAGCTTTTGGGGTTATTTACGAAAACATGGATGAAC

TGATGGCCGCTTACAATAAAGTGCCTAACTCAATCTCCTTCGA

GAACGCGCTGAAGATGCGTCTTAATGCCGTTACAAGCACCAAT

ACAGAAGGGGCTGTTATCCACATTAATCGCAGTTGTAAGCTGT

GGTCAGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAGGA

GACGGGGATCCCTGTTGTTTCGTTCGACGGAGATCAAGCGGAT

CCCCGTAACTTCTCCGAGGCTCAATATGACACTCGCATCCAAG

GTTTAAATGAGGTGATGGTCGCGAAAAAAGAAGCAGAGTGAG

CTTtaagaaggagatatacatATGTCGAATAGTGACAAGTTTTTTAACGA

CTTCAAGGACATTGTGGAAAACCCAAAGAAGTATATCATGAA

GCATATGGAACAAACGGGACAAAAAGCCATCGGTTGCATGCC

TTTATACACCCCAGAAGAGCTTGTCTTAGCGGCGGGTATGTTT

CCTGTTGGAGTATGGGGCTCGAATACTGAGTTGTCAAAAGCCA

AGACCTACTTTCCGGCTTTTATCTGTTCTATCTTGCAAACTACT

TTAGAAAACGCATTGAATGGGGAGTATGACATGCTGTCTGGTA

TGATGATCACAAACTATTGCGATTCGCTGAAATGTATGGGACA

AAACTTCAAACTTACAGTGGAAAATATCGAATTCATCCCGGTT ACGGTTCCACAAAACCGCAAGATGGAGGCGGGTAAAGAATTT

CTGAAATCCCAGTATAAAATGAATATCGAACAACTGGAAAAA

ATCTCAGGGAATAAGATCACTGACGAGAGCTTGGAGAAGGCT

ATTGAAATTTACGATGAGCACCGTAAAGTCATGAACGATTTCT

CTATGCTTGCGTCCAAGTACCCTGGTATCATTACGCCAACGAA

ACGTAACTACGTGATGAAGTCAGCGTATTATATGGACAAGAA

AGAACATACAGAGAAGGTACGTCAGTTGATGGATGAAATCAA

GGCCATTGAGCCTAAACCATTCGAAGGAAAACGCGTGATTAC

CACTGGGATCATTGCAGATTCGGAGGACCTTTTGAAAATCTTG

GAGGAGAATAACATTGCTATCGTGGGAGATGATATTGCACAC

GAGTCTCGCCAATACCGCACTTTGACCCCGGAGGCCAACACAC

CTATGGACCGTCTTGCTGAACAATTTGCGAACCGCGAGTGTTC

GACGTTGTATGACCCTGAAAAAAAACGTGGACAGTATATTGTC

GAGATGGCAAAAGAGCGTAAGGCCGACGGAATCATCTTCTTC

ATGACAAAATTCTGCGATCCCGAAGAATACGATTACCCTCAGA

TGAAAAAAGACTTCGAAGAAGCCGGTATTCCCCACGTTCTGAT

TGAGACAGACATGCAAATGAAGAACTACGAACAAGCTCGCAC

CGCTATTCAAGCATTTTCAGAAACCCTTTGACGCTtaagaaggagatat acatATGTTCTTTACGGAGCAACACGAACTTATTCGCAAACTGGC

GCGTGACTTTGCCGAACAGGAAATCGAGCCTATCGCAGACGA

AGTAGATAAAACCGCAGAGTTCCCAAAAGAAATCGTGAAGAA

GATGGCTCAAAATGGATTTTTCGGCATTAAAATGCCTAAAGAA

TACGGAGGGGCGGGTGCGGATAACCGCGCTTATGTCACTATTA

TGGAGGAAATTTCACGTGCTTCCGGGGTAGCGGGTATCTACCT

GAGCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTTATTGGTC

GGAACCGATGAGCAAAAAGAAAAGTACCTTAAGCCTATGATC

CGCGGCGAGAAGACTCTGGCGTTCGCCCTGACAGAGCCTGGT

GCTGGCTCTGATGCGGGTGCGTTGGCTACTACTGCCCGTGAAG

AGGGCGACTATTATATCTTAAATGGCCGCAAGACGTTTATTAC

AGGGGCTCCTATTAGCGACAATATTATTGTGTTCGCAAAAACC

GATATGAGCAAAGGGACCAAAGGTATCACCACTTTCATTGTG

GACTCAAAGCAGGAAGGGGTAAGTTTTGGTAAGCCAGAGGAC

AAAATGGGAATGATTGGTTGTCCGACAAGCGACATCATCTTGG

AAAACGTTAAAGTTCATAAGTCCGACATCTTGGGAGAAGTCA

ATAAGGGGTTTATTACCGCGATGAAAACACTTTCCGTTGGTCG

TATCGGAGTGGCGTCACAGGCGCTTGGAATTGCACAGGCCGC

CGTAGATGAGGCGGTAAAGTACGCCAAGCAACGTAAACAATT

CAATCGCCCAATCGCGAAATTTCAGGCCATTCAATTTAAACTT

GCCAATATGGAGACTAAATTAAATGCCGCTAAACTTCTTGTTT

ATAACGCAGCGTACAAAATGGATTGTGGAGAAAAAGCCGACA

AGGAAGCCTCTATGGCTAAATACTTTGCTGCTGAATCAGCGAT

CCAAATCGTTAACGACGCGCTGCAAATCCATGGCGGGTATGG

CTATATCAAAGACTACAAGATTGAACGTTTGTACCGCGATGTG

CGTGTGATCGCTATTTATGAGGGCACTTCCGAGGTCCAACAGA

TGGTTATCGCGTCCAATCTGCTGAAGTAATACTtaagaaggagatatac atATGAAAATCTTGGCATACTGCGTCCGCCCAGACGAGGTAGA

CTCCTTTAAGAAATTTAGTGAAAAGTACGGGCATACAGTTGAT

CTTATTCCAGACTCTTTTGGACCTAATGTCGCTCATTTGGCGAA

GGGTTACGATGGGATTTCTATTCTGGGCAACGACACGTGTAAC CGTGAGGCACTGGAGAAGATCAAGGATTGCGGGATCAAATAT

CTGGCAACCCGTACAGCCGGAGTGAACAACATTGACTTCGAT

GCAGCAAAGGAGTTCGGTATTAACGTGGCTAATGTTCCCGCAT

ATTCCCCCAACTCGGTCAGCGAATTTACCATTGGATTGGCATT

AAGTCTGACGCGTAAGATTCCATTTGCCCTGAAACGCGTGGAA

CTGAACAATTTTGCGCTTGGCGGCCTTATTGGTGTGGAATTGC

GTAACTTAACTTTAGGAGTCATCGGTACTGGTCGCATCGGATT

GAAAGTGATTGAGGGCTTCTCTGGGTTTGGAATGAAAAAAAT

GATCGGTTATGACATTTTTGAAAATGAAGAAGCAAAGAAGTA

CATCGAATACAAATCATTAGACGAAGTTTTTAAAGAGGCTGAT

ATTATCACTCTGCATGCGCCTCTGACAGACGACAACTATCATA

TGATTGGTAAAGAATCCATTGCTAAAATGAAGGATGGGGTATT

TATTATCAACGCAGCGCGTGGAGCCTTAATCGATAGTGAGGCC

CTGATTGAAGGGTTAAAATCGGGGAAGATTGCGGGCGCGGCT

CTGGATAGCTATGAGTATGAGCAAGGTGTCTTTCACAACAATA

AGATGAATGAAATTATGCAGGATGATACCTTGGAACGTCTGA

AATCTTTTCCCAACGTCGTGATCACGCCGCATTTGGGTTTTTAT

ACTGATGAGGCGGTTTCCAATATGGTAGAGATCACACTGATGA

ACCTTCAGGAATTCGAGTTGAAAGGAACCTGTAAGAACCAGC GTGTTTGTAAATGA

[01222] In some embodiments, the genetically engineered bacteria

comprise a sequence which has at least about 80% identity with one or more sequences of Table 46. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table

46. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 46. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 46. In another

embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 46. Accordingly, In some embodiments, the genetically

engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%,

83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, or 99% identity with one or more sequences of Table 46. In another embodiment, the genetically engineered bacteria comprise one or more sequence of Table 46. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 46.

[01223] In some embodiments, the genetically engineered bacteria

comprise a sequence which has at least about 80% identity with SEQ ID NO: 166. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 166. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 166. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 166. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 166. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 166. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 166. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 166.

[01224] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 168. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 168. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 168. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 168. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 168. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 168. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 168. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 168.

[01225] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 170. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 170. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 170. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 170. In another embodiment, the bcd.2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 170. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 170. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 170. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 170.

[01226] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 172. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 172. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 172. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 172. In another embodiment, the bcd.2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 172. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 172. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 172. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 172.

[01227] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 174. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 174. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 174. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 174. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 174. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 174. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 174. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 174.

[01228] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 178. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 178. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 178. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 178. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 178. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 178. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 178. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 178.

[01229] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 184. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 184. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 184. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 184. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 184. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 184. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 184. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 184.

[01230] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 186. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 186. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 186. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 186. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 186. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 186. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 186. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 186.

[01231] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 192. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 192. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 192. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 192. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 192. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 192. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 192. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 192. [01232]

Example 21. Tryptophan production in an engineered strain of E. coli Nissle

[0170] A number of tryptophan metabolites, either host-derived (such as tryptamine or kynurerine) or intestinal bacteria-derived (such as indoleacetate or indole), have been shown to downregulate inflammation in the context of IBD, via the activation of the AhR receptor. Other tryptophan metabolites, such as the bacteria- derived indolepropionate, have been shown to help restore intestinal barrier integrity, in experimental models of colitis. In this example, the E. coli strain Nissle was engineered to produce tryptophan, the precursor to all those beneficial metabolites.

[0171] First, in order to remove the negative regulation of tryptophan biosynthetic genes mediated by the transcription factor TrpR, the trpR gene was deleted form the E. coli Nissle genome. The tryptophan operon trpEDCBA was amplified by PCR from the E. coli Nissle genomic DNA and cloned in the low-copy plasmid pSClOl under the control of the tet promoter, downstream of the tetR repressor gene. This tet- trpEDCBA plasmid was then transformed into the AtrpR mutant to obtain the AtrpR, tet- trpEDCBA strain. Subsequently, a feedback resistant version of the aroG gene (aroG^ ) from E. coli Nissle, coding for the enzyme catalyzing the first committing step towards aromatic amino acid production, was synthetized and cloned into the medium copy plasmid pl5A, under the control of the tet promoter, downstream of the tetR repressor. This plasmid was transformed into the AtrpR, tet-trpEDCBA strain to obtain the AtrpR, tet-trpEDCBA, tet-aroG^ strain. Finally, a feedback resistant version of the tet- trpEBCDA construct (tet-trpE^BCDA) was generated from the tet-trpEBCDA. Both the tet-aroG^ and the tet-trpE^' BCDA constructs were transformed into the AtrpR mutant to obtain the AtrpR, tet-trpE^DCBA, tet-aroG^fbr strain.

[0172] All generated strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3mL LB with antibiotics in culture tubes. After two hours of growth at 37C at 250rpm, lOOng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3mL M9 minimal media. Cells were spun down again at 4,000rpm for 5min, resuspended in 3mL M9 minimal media with 0.5% glucose and placed at 37C at 250rpm. 200uL were collected at 2h, 4h and 16h and tryptophan was quantified by LC- MS/MS in the bacterial supernatant. FIG. 44A shows that tryptophan is being produced and secreted by the AtrpR, tet-trpEDCBA, tet-aroCf^b strain. The production of tryptophan is significantly enhanced by expressing the feedback resistant version of trpE.

Example 52. Improved tryptophan production by using a non-PTS carbon source and by deleting the tnaA gene encoding for the tryptophanase enzyme converting tryptophan into indole

[0173] One of the precursor molecule to tryptophan in E. coli is

phosphoenolpyruvate (PEP). Only 3% of available PEP is normally used to produce aromatic acids (that include tryptophan, phenylalanine and tyrosine). When E. coli is grown using glucose as a sole carbon source, 50% of PEP is used to import glucose into the cell using the phosphotransferase system (PTS). In order to increase tryptophan production, a non-PTS oxidized sugar, glucuronate, was used to test tryptophan secretion by the engineered E. coli Nissle strain AtrpR, tet-trpE^DCBA, tet-aro '^r. In addition, the tnaA gene, encoding the tryptophanase enzyme, was deleted in the AtrpR, tet-trpE^DCBA, tet-aro '^r strain in order to block the conversion of tryptophan into indole to obtain the AtrpRAtnaA, tet-trpE^DCBA, tet-aro '^r strain.

[0174] t e AtrpR, tet-trpE^DCBA, tet-aroCf^br and AtrpRAtnaA, tet- trpE^DCBA, tet-aro '^r strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3mL LB with antibiotics in culture tubes. After two hours of growth at 37C at 250rpm, lOOng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3mL M9 minimal media. Cells were spun down again at 4,000rpm for 5min, resuspended in 3mL M9 minimal media with 1% glucose or 1% glucuronate and placed at 37C at 250rpm or at 37C in an anaerobic chamber. 200uL were collected at 3h and 16h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 22B shows that tryptophan production is doubled in aerobic condition when the non- PTS oxidized sugar glucoronate was used. In addition, the deletion of tnaA had a positive effect on tryptophan production at the 3h time point in both aerobic and anaerobic conditions and at the 16h time point, only in anaerobic condition. Example 22. Improved tryptophan production by increasing the rate of serine biosynthesis in E. coli Nissle

[0175] The last step in the tryptophan biosynthesis in E. coli consumes one molecule of serine. In this example, we demonstrate that serine availability is a limiting factor for tryptophan production and describe the construction of the tryptophan producing E. coli Nissle strains AtrpRAtnaA, tet-trpE^DCBA, tet-aroG^' serA and AtrpRAtnaA, tet-trpE^ DCBA, tet-aroG^fbrserA^Jbr strains.

[0176] the AtrpRAtnaA, tet-trpE^DCBA, tet-aro ^r strain was grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3mL LB with antibiotics in culture tubes. After two hours of growth at 37C at 250rpm, lOOng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3mL M9 minimal media. Cells were spun down again at 4,000rpm for 5min, resuspended in 3mL M9 minimal media with 1% glucuronate or 1% glucuronate and lOmM serine and placed at 37C an anaerobic chamber. 200uL were collected at 3h and 16h and tryptophan was quantified by LC- MS/MS in the bacterial supernatant. FIG. 22C shows that tryptophan production is improved three fold by serine addition.

[0177] In order to increase the rate of serine biosynthesis in the

AtrpRAtnaA, tet-trpE^ DCBA, tet-aro '^r strain, the serA gene from E. coli Nissle encoding the enzyme catalyzing the first step in the serine biosynthetic pathway was amplified by PCR and cloned into the tet-aro '^r plasmid by Gibson assembly. The newly generated tet-aroCf^br-serA construct was then transformed into a AtrpRAtnaA, tet- trpE^DCBA strain to generate the AtrpRAtnaA, tet-trpE^DCBA, tet-aro ^r -serA strain. The tet- aro " ^r-serA construct was further modified to encode a feedback resistant version of serA (serA^ ). The newly generated tet-aro '¹ -serA^ construct was used to produce the AtrpRAtnaA, tet-trpE^DCBA, tet-aro '^r -serA^^r strain, optimized to improve the rate of serine biosynthesis and maximize tryptophan production.

Example 23. Comparison of Various Tryptophan Producing Strains

[0178] Compare the rates of tryptophan production in the different strains generated, the following constructs and strains were generated according to methods and sequences described herein, and assayed for tryptophan production in the presence of glucuronate as a carbon source under aerobic conditions. SYN2126 comprises AtrpRAtnaA (AtrpRAtnaA). SYN2323 comprises AtrpRAtnaA and a tetracycline inducible construct for the expression of feedback resistant aroG on a plasmid

(AtrpRAtnaA, tet-aroGfbr). SYN2339 comprises AtrpRAtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (AtrpRAtnaA, tet-aroGfbr, tet- trpEfbrDCBA). SYN2473 comprises AtrpRAtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG and SerA on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (AtrpRAtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA). SYN2476 comprises AtrpRAtnaA and a tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a plasmid (AtrpRAtnaA, tet-trpEfbrDCBA).

[0179] Overnight cultures were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37C, 250rpm). Next, cells were induced with lOOng/mL ATC for 2 hours (37C, 250rpm), spun down, washed with cmL M9, spun down again and resuspended in 3mL M9+l% glucuronate. Cells were plated for CFU counting. For the assay, the cells were placed af 37C with shaking at 250rpm. Supernatants were collected at lh, 2h, 3h, 4h 16h for HPLC analysis for tryptophan. As seen in FIG. 22D, results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.

Example 24. Bacterial Production of Indole Acetic Acid (IAA)

[0180] The ability of a strain comprising tryptophan production circuits and additionally Indole-3-pyruvate decarboxylase from Enterobacter cloacae (IpdC) and Indole- 3 -acetaldehyde dehydrogenase from Ustilago maydis (Iadl) to produce indole acetic acid (IAA) was tested. The following strains were generated according to methods described herein and tested.

[0181] SYN2126: comprises AtrpR and AtnaA (AtrpRAtnaA). SYN2339 comprises circuitry for the production of tryptophan; AtrpR and AtnaA, a first tetracline inducible trpEfbrDCBA construct on a first plasmid(pSClOl), and a second tetracycline inducible aroGfbr construct on a second plasmid (AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr (pl5A)). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises trpDH- ipdC-iadl incorporated at the end of the second construct (AtrpRAtnaA, tetR-Ptet- trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr-trpDH-ipdC-iadl (pl5A)).

[0182] Overnight cultures of the strains were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37C, 250rpm). Strains were then induced with lOOng/mL ATC for 2 hours (37C, 250rpm). Cells were spun down, and resuspended in lmL M9+l% glucuronic acid and CFUs were quantified CFUs using the cello meter. Supernatants were collected at lh, 2.5h and 18h for LCMS analysis of tryptophan and indole acetic acid as described herein.

[0183] As seen in FIG. 23A, SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 containing the additional IAA producing circuitry produces amounts of IAA that are comparable to the amounts of tryptophan produced in its parent SYN2339. No tryptophan is measured, indicating that all tryptophan produced in SYN2342 is efficiently converted into IAA.

Example 25. Tryptamine Production Comparing Two Tryptophan Decarboxylases

[0184] The efficacy of two tryptophan decarboxylases (tdc), one from

Catharanthus roseus (tdcc_r)and a second from Clostridium sporogenes (tdcc_s) in producing tryptamine from tryptophan was tested. The following strains were generated according to methods described herein and tested.

[0185] SYN2339 comprises AtrpR and AtnaA and a tetracycline inducible trpE fbr DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG^fcr on a second plasmid (AtrpRAtnaA,

(pSClOl), fbr

tetR-Ptet-aroG^luI (pl5A)). SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine. SYN2340 comprises AtrpR and AtnaA and a tetracycline inducible trpE fbr DCBA construct on a plasmid and another tetracycline fb

inducible construct expressing aroG tdcc_r on a second plasmid (AtrpRAtnaA, tetR-P_tet- trpE DCBA (pSClOl), tetR-P_tet-aroG -tdc_Cr (pl5A)). SYN2794 comprises AtrpR and

AtnaA and a tetracycline inducible trpE fbr DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG fbr tdcc_s on a second plasmid

(AtrpRAtnaA,

(pl5A)).

[0186] Overnight cultures of the strains were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37C, 250rpm). Strains were then induced with lOOng/mL ATC for 2 hours (37C, 250rpm). Cells were spun down, and resuspended in lmL M9+l% glucuronic acid and CFUs were quantified CFUs using the cello meter. Supernatants were collected at 3h and 18h for LCMS analysis of tryptophan and tryptamine, as described herein.

[0187] As seen in FIG. 23B, Tdcc_s from Clostridium sporogenes is more efficient than Tdcc_r from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine.

Example 26. Kynurenine quantification in bacterial supernatant by LC-MS/MS

Sample Preparation

[0188] Kynurenine standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μg/mL) were prepared in water from Kynurenine stock in 0.5N HC1. Sample (10 μί)^ηά standards) were mixed with 90 of ACN/H₂0 (60:30, v/v) in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil and mixed well, and centrifuged at 4000rpm for 5min. ΙΟμΙ_^ of the solution was transferred to a round-bottom 96-well plate, and 90 uL 0.1% formic acid in water was added to the sample. The plate was heat sealed with a ClearASeal sheet and mixed well.

LC-MS/MS method

[0189] Kynurenine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 47, Table 48, and Table 49 provide the summary of the LC-MS/MS method.

Table 47. LC-MS/MS Method

Mobile Phase A: 99.9% H20, 0.1%

Formic Acid

Mobile Phase B: 99.9% ACN, 0.1%

Formic Acid

Injection volume: 10uL

Table 48. HPLC Method

Table 49. Tandem Mass Spectrometry

Example 27. Kynurenine quantification in tumor tissue by LC-MS/MS

Sample Preparation

[0190] Kynurenine standards (100, 20, 4, 0.8, 0.16, 0.032, 0.0064 μg/mL) were prepared in water from Kynurenine stock in 0.5N HC1. Weighed tumor tissues were homogenized with PBS in BeadBug prefilled tubes using a FastPrep homogenizer and the homogenate was transferred into a V-bottom 96-well plate and centrifuged at 4000rpm for lOmin. Sample (40

of ACN containing \\aglmL of Adenosine- 13C₅ (used as internal standard) in the final solution in a V-bottom 96-well plate.. The plate was heat-sealed with a AlumASeal foil and mixed well, and centrifuged at 4000rpm for 5min. ΙΟμΙ_^ of the solution was transferred to a round-bottom 96-well plate, and 90 uL 0.1% formic acid in water was added to the sample. The plate was heat sealed with a ClearASeal sheet and mixed well.

LC-MS/MS method

[0191] Kynurenine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 50, Table 51, and Table 52 provide the summary of the LC-MS/MS method.

Table 50. LC-MS/MS Method

Table 51. HPLC Method:

Table 52. Tandem Mass Spectrometry

SRM transitions:

Kynurenine: 209.1 /91 .2

209.1/146.1

Adenosine- 13C₅: 273.1 /1 36.2

Example 28. Tryptophan and Anthranilic acid quantification in bacterial supernatant by LC-MS/MS

Sample Preparation

[0192] Tryptophan and Anthranilic acid stock (10 mg/mL) were prepared in

0.5N HCl and aliquoted in 1.5 mL microcentrifuge tubes (100

Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μg/mL) of each were prepared in water. Sample (10

(and standards) were mixed with 90

of Tryptophan-d5 in the final solution in a V-bottom 96-well plate. The plate was heat- sealed with a AlumASeal foil, mixed well, and centrifuged at 4000rpm for 5ητίη.10μί of the solution was transferred into a round-bottom 96-well plate and 90 uL 0.1% formic acid in water was added to the sample. The plate was heat-sealed with a

ClearASeal sheet and mixed well.

LC-MS/MS method

[0193] Tryptophan and Anthranilic acid were measured by liquid

chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 53 Table 54, and

Table 55 provide the summary of the LC-MS/MS method.

Table 53. LC-MS/MS Method

Table 54. HPLC Method

Table 55. Tandem Mass Spectrometry

Example 29. Tryptophan and Anthranilic acid quantification in tumor tissue by LC-MS/MS

Sample Preparation

[0194] Tryptophan and Anthranilic acid stock (10 mg/mL) were prepared in

0.5N HCl and aliquoted in 1.5 mL microcentrifuge tubes (100

Standards (100, 20, 4, 0.8, 0.16, 0.032, 0.0064 μg/mL) of each were prepared in water. Weighed tumor tissues were homogenized with PBS in BeadBug prefilled tubes using a FastPrep homogenizer. The homogenate was transferred into a V-bottom 96-well plate and centrifuged at 4000rpm for lOmin. 40

of sample (and standards) was mixed with 60

of Tryptophan-d5 in the final solution in a V-bottom 96- well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000rpm for lOmin. ΙΟμΙ_^ of the solution was transferred into a round- bottom 96-well plate, and 90 uL 0.1% formic acid in water was added to the sample. The plate was heat-sealed with a ClearASeal sheet and mixed well. LC-MS/MS method

[0195] Tryptophan and Anthranilic acid were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 56 Table 57, and Table 58 provide the summary of the LC-MS/MS method.

Table 56. LC-MS/MS Method

Table 57. HPLC Method

Table 58. Tandem Mass Spectrometry

Example 30. Quantification of Tryptamine in Bacterial Supernatant by Liquid Chromatography-Mass Spectrometry (LC-MS)

[0196] Tryptamine acid stock (10 mg/mL) were prepared in 0.5N HC1, aliquoted in 1.5 niL microcentrifuge tubes (100 μί), and stored at -20°C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μg/mL) were prepared. Samples (10 L) and standards were mixed with 90 μΐ_^ of ACN/H20 (60:30, v/v) containing ^g/mL of tryptamine-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000rpm for 5min. The solution (ΙΟμΙ_^) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.

LC-MS/MS method

[0197] Tryptamine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 59., Table 60, and Table 61 provide the summary of the LC- MS/MS method.

Table 59. HPLC Method

Table 60. HPLC Method:

Table 61. Tandem Mass Spectrometry Ion Source HESI-II

Polarity Positive

SRM transitions

Tryptamine 161.1/144.1

Example 46. Quantification of Tryptophan, Indole-3-acetate, Indole-3-lactate, Indole-3-propionate in Bacterial Supernatant by High-pressure Liquid

Chromatography (HPLC)

[0198] Samples were thawed on ice and centrifuged at 3,220 x g for 5min at

4°C. 80μί of the supernatant was pipetted, mixed with 20μί 0.5% formic acid in water, and analyzed by HPLC using a Shimadzu Prominence-I. HPLC conditions used for the quantification of tryptophan, indole- 3 -acetate, indole-3-lactate and indole-3-propionate are described in Table 67.

Table 62. HPLC Analysis

Spectrum resolution 512

Slit Width 8 nm

Compound Wavelength (nm) Retention time (min)

Tryptophan 274 1.3

Indole- 3 -acetate 274 3.5

Indole- lactate 274 3.3

Indo le- 3 -propionate 274 3.7

Example 31. Efficacy of Tryptamine and IAA-Expressing Bacteria in a Mouse Model of IBD and mouse model of Obesity/T2DM

[0199] Bacteria harboring the cassette expressing are grown overnight in LB.

Bacteria are then diluted 1: 100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by

centrifugation.

[0200] For the study, Strain SYN2342 harboring the tryptophan production and or indole acetic acid production gene cassette as described (Example 24) is modified (SYN2342*). Additional strain SYN2794 harboring tryptamine production circuitry is modified (SYN2794*). In both strains, the tet inducible promoter is replaced with an FNR promoter, and constructs are integrated into the bacterial chromosome according to methods described herein. In described above are grown overnight in LB. Bacteria are then diluted 1: 100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5. Bacteria comprising a the FNR- inducible promoter are induced in LB at 37C for up to 4 hours in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N2, 5% C02, 5%H2, and 20mM nitrate) at 37° C and then pelleted by centrifugation.

[0201] To analyze the efficacy of the bacteria in vivo, bacteria are resuspended in phosphate buffered saline (PBS) and 100 microliters is administered by oral gavage to mice daily for 8 weeks. Alternatively, the bacteria can be supplemented in the drinking water (5 x 10⁹ CFU bacteria/mL).

[01233] At Day 0, 40 C57BL6 mice (8 weeks of age) are weighed and randomized into the following five treatment groups (n=8 per group): H₂0 control (group 1); 0.5% DSS control (group 2); 0.5% DSS + 100 mM indole (group 3); 0.5% DSS + wild type Nissle (group 4); and 0.5% DSS + SYN2794* (group 5). and 0.5% DSS + SYN2342* (group 6). After randomization, the cage water for group 3 is changed to water supplemented with butyrate (100 mM), and groups 4, 5, and 6 are administered 100 μΐ. of SYN94, SYN2794* and SYN2342* by oral gavage,

respectively. At Day 1, groups 4, 5, and 6 are gavaged with bacteria in the morning, weighed, and gavaged again in the evening. Groups 4, 5, and 6 are also gavaged once per day for Day 2 and Day 3.

[01234] At Day 4, groups 4, 5, and 6 are gavaged with bacteria, and then all mice are weighed. Cage water is changed to either H₂0 + 0.5% DSS (groups 2, 4, and 5), or H₂0 + 0.5% DSS supplemented with 100 mM butyrate (group 3). Mice from groups 4, 5, and 6 are gavaged again in the evening. On Days 5-7, groups 4, 5, and 6 are gavaged with bacteria in the morning, weighed, and gavaged again in the evening.

[01235] At Day 8, all mice are fasted for 4 hours, and groups 4, 5, and 6 are gavaged with bacteria immediately following the removal of food. All mice are then weighed, and gavaged with a single dose of FITC-dextran tracer (4 kDa, 0.6 mg/g body weight). Fecal pellets are collected; however, if colitis is severe enough to prevent feces collection, feces are harvested after euthanization. All mice are euthanized at exactly 3 hours following FITC-dextran administration. Animals are then cardiac bled and blood samples are processed to obtain serum. Levels of mouse lipocalin 2, calprotectin, and CRP-1 are quantified by ELISA, and serum levels of FITC-dextran are analyzed by spectrophotometry (e.g., as described in co-owned pending International Patent

Application PCT/US2016/050836, the contents of which is herein incorporated by reference in its entirety). Distal colonic sections are fixed and scored for inflammation and ulceration. Colonic tissue is homogenized and measurements are made for myeloperoxidase activity using an enzymatic assay kit and for cytokine levels (IL-Ιβ, TNF-a, IL-6, IFN-γ and IL-10).

[0202] In a second study, the genetically engineered bacteria comprising a tryptamine cassette or a IAA production cassette and a FNR-butyrate cassette are assessed in this model. Suitable butyrate cassettes are described in International Patent Application PCT/US2016/20530 (published as WO/2016/141108) and International Patent Application PCT/US2016/050836, and US Patent Application 15/260,319, the contents of each of which is herein incorporated by reference in its entirety.

[0203] In a second study, SYN2794 and SYN2342 are assessed in a T2D model (ZFR fatty rats). [0204] The ability of engineered strain S YN2794 and S YN2342 to produced tryptamine and IAA and to improve glucose homeostasis is tested in vivo in the Zucker -fatty rat (ZFR) model (e.g., as described in She et al., Leucine and Protein Metabolism in Obese Zucker Rats; PLoS One. 2013; 8(3): e59443; and She et al., Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched chain amino acid (BCAA) metabolism, Am J Physiol Endocrinol Metab. 2007 December ; 293(6): E1552-E1563, the contents of which are herein incorporated by reference in its entirety). SYN2794 and SYN2342 are compared to wild type Nissle with a streptomycin resistance in this study.

[01236] To prepare the cells for this study, bacterial growth and induction conditions are as follows. Overnight cultures (5mL) with Strep (control Nissle) or Carbenicillin (SYN1980). 500mL LB flasks are inoculated with the overnight cultures., and grown for 2h at 37C with 250rpm. Next tetracycline (ATC lOOng/mL) is added for 2 hours. Cultures are spun down at 4C for 30min, at 4,000 rpm and the pellets are resuspended in lOmL formulation buffer (PBS, 15% glycerol, 2g/L glucose, 3mM thymidine), aliquoted in 2 ml cryovials and kept at -80C.

[01237] Zucker fatty rats are purchased from Charles River Laboratory. Animals are housed with an ambient temperature of 21 to 23°C on a 12-hour light/dark cycle and given free access to water and a rodent chow diet (Harland Teklad 2018, Madison, Wisconsin; diet contains protein (18%) and leucine (1.8%)).

[01238] On day -7, animals (Zucker fatty rats (ZFR) 11 weeks of age) are given free access to water and a rodent chow diet (Harland Teklad 2018, Madison, Wisconsin; diet contains protein (18%) and leucine (1.8%). On day 1, animals are randomized into treatment groups. Mice are bled and and urine is collected (T=0). Rats are grouped as follows: Group 1: H20 (n=10); Group 2: wild type Nissle with streptomycin resistance (n=10); Group 3: SYN2794* (n=10) and Group 4: SYN2342* strain (n=10); For Groups 2 ,3, and 4, rats are gavaged with le9 CFUs/dose in 250 ul/dose in the am and pm (2 doses per day), and ATC (20 ng/mL) and 5% sucrose is added to the drinking water. Group 1 is dosed with 250 ul H20. Mice are continued on the same rodent chow with 18% protein, 1.8% leucine throughout the study.

[01239] On day 2, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or H20 (Group 1). On day 3, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or water, and animals are weighed, blood is drawn and collected and urine is collected at 1 hour post last dose and stored on ice for LC/MS analysis. On days 4-6, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or H20 (Group 1). On day 7, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or water, and animals are weighed, blood is drawn and collected and urine is collected at 1 hour post last dose and stored on ice for LC/MS analysis. On day 8- 13, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or H20 (Group 1). On day 14, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or water, and animals are weighed, blood is drawn and collected and urine is collected at 1 hour post last dose and stored on ice for LC/MS analysis. Animals are sacrificed and tissues (liver and muscle) are extracted, ground in 1 niL 10% ACN to test metabolite levels are stored on ice for LC/MS analysis. Stool sampleas are collected andtryptophan, IAA, and tryptamine levels are measured by LC/MS. Additionally, a set of key clinical analytes are measured by non- MS methods (glucose, insulin, total ketones, delta-OH-butyrate, lactate, non-essential fatty acids and triglicerides). Triglyceride content is measured in liver, muscle, adipose and heart tissues, along with other related metabolites, as described herein and known in the art.

[01240] This study determines whether the genetically engineered strain can positively affect blood glucose levels, improve glucose homeostasis and/or reduce insulin resistance, and decrease liver fat.

Example 32. Efficacy of IDO-Expressing Bacteria in a Mouse Model of

Rheumatoid Arthritis

[0205] Bacteria harboring the cassette expressing IDO are grown overnight in

LB. Bacteria are then diluted 1 : 100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by

centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters is administered by oral gavage to mice.

[0206] To induce arthritis, all mice were immunized intradermally in two sites at the base of the tail with 200 μg of bovine, chicken, or mouse type II collagen in complete Freund's adjuvant (CFA) as described (Inglis et al, Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen; Arthritis Res Ther. 2007; 9(5): R113). To prepare the CFA, 100 mg of desiccated killed Mycobacterium tuberculosis H37Ra (BD Biosciences) was ground with a pestle and mortar to produce a fine powder and then suspended in 30 niL of incomplete Freund's adjuvant (BD Biosciences).

[0207] For macroscopic assessment of arthritis, the thickness of each affected hind paw was measured daily with microcalipers (Kroeplin GmbH, Schliichtern, Germany) and the diameter was expressed as an average for inflamed hind paws per mouse. Animals were also scored for clinical signs of arthritis as follows: 0 = normal, 1 = slight swelling and/or erythema, 2 = pronounced oedematous swelling, and 3 = joint rigidity. Each limb was graded, allowing a maximum score of 12 per mouse. After completion of the experiment, mice were sacrificed and hind paws were immersion- fixed in 10% (vol/vol) buffered formalin and decalcified with 5.5% EDTA

(ethylenediaminetetraacetic acid) in buffered formalin.

[0208] For histological assessment of arthritis, arthritic mice were killed up to

2 weeks after disease onset (early arthritis, n = 8) or 6 to 8 weeks following onset (late arthritis, n = 8). Joints were decalcified and paraffin-embedded, and sections (10 μιη) were stained (haematoxylin and eosin) for conventional histology. Joints were classified according to the presence or absence of inflammatory cell infiltrates (defined as focal accumulations of leukocytes).

[0209] To analyze T-cell activity, inguinal lymph nodes were excised from mice with early or late arthritis. Lymph node cells (LNCs) were cultured in RPMI 1640 containing foetal calf serum (10% vol/vol), 2-mercaptoethanol (20 μΜ), L-glutamine (1% wt/vol), penicillin (100 U/mL), and streptomycin (100 μg/mL) in the presence or absence of type II collagen or the synthetic collagen fragment CII256-270 (both at 50 μg/mL). After 48 hours, 100 μΐ_^ of culture medium was carefully removed for measurement of cytokines and the remaining cells were pulsed with 1 μθ ³H thymidine per well for a further 18 hours. Cells were then harvested and plates were assessed for ³H thymidine incorporation. Each assay was performed on a minimum of three occasions. Secreted interferon-gamma (IFN-γ), interleukin (IL)-5, and IL-10 were measured in the culture supernatant by sandwich ELISA using capture and detection antibody pairs (BD Biosciences). [0210] In another study, the ability of engineered strain SYN2794 and

SYN2342 to produced tryptamine and IAA and to improve measurements relating to arthritis are assessed.

Example 33. Evaluation of anti-tumor efficacy using bacteria engineered to express Kynureninase in a breast cancer model and a melanoma model

[0211] In this study the eveloved strains derived from SYN2027 and SYN2028 are tested in a breat cancer and a melanoma model. Six to eight week old female Balb/c mice are implanted as described above, implanted subcutaneously with 4T1 s.c. tumors and in a second study wityh B 16 melanoma tumors into the right flank of each animal (BalbC/J (female, 8 weeks)), and tumor growth is monitored for approximately 10 days. When the tumors reach about -100- 150 mm3, animals are randomized into groups for bacterial dosing.

[0212] Treatment is initiated, and mice are intratumorally administered either

E. coli Nissle engineered to express anti-PD- 1 antibody and kynureninase, or the control bacteria (E. coli Nissle 1917) 2 to 3 times a week at the approporate dose deterimined above for 21-28 days. A another control group is treated systemically with PD- 1 antibody. Another group is treated systemically with an anti-PDl antibody and interatumorally with the kynureninase producing strains. A control group is injected with the same volume of PBS. Intratumoral injections are performed as described above. As another control group for benchmarking, a concurrent administration of anti- PD- 1 antibody and IDO inhibitor (e.g., indoximod) is performed in a separate treatment group. A corresponding human IgG control is administered in parallel. Anti-CTLA4 antibody, human immunoglobulin (hlgG) vehicle control and IDO inhibitor are administered intraperitoneally.

[0213] As described above, the animals are weighed, and tumor growth is assessed. Statistical significance is tested according to methods known in the art.

[0214] Animals are euthanized at the end of the study or when tumors reach

2000 mm3 (or before if it is deemed that tumors are adversely affecting animal health).

Example 34. Kynurenine consuming strains decrease tumoral kynurenine levels in the CT26 murine tumor model [0215] The ability of genetically engineered bacteria comprising kynureninase from Pseudomonas fluorescens to consume kynurenine in vivo in the tumor

environment was assessed. SYN1704, an E. coli Nissle strain comprising a deletion in Trp:E and a medium copy plasmid expressing kynureninase from Pseudomonas fluorescens under control of a constitutive promoter (Nissle delta TrpE::CmR +

Pconstitutive-Pseudomonas KYNU KanR) was used.

[0216] In both studies, CT26 cells obtained from ATCC were cultured according to guidlelines provided. Approximately -lxlO⁶ cells/mouse in PBS were implanted subcutaneously into the right flank of each animal (BalbC/J (female, 8 weeks)), and tumor growth was monitored for approximately 10 days. When the tumors reached about ~100-150mm , animals were randomized into groups for dosing.

[0217] For intratumoral injection, bacteria were grown in LB broth until reaching an absorbance at 600 nm (A600 nm) of 0.4 (corresponding to 2 X 10 colony- forming units (CFU)/mL) and washed twice in PBS. The suspension was diluted in PBS or saline so that 100 uL can be injected at the appropriate doses intratumorally into tumor-bearing mice.

[0218] Approximately 10 days after CT 26 implantation, bacteria were suspended in 0.1 mL of PBS and mice were injected (le7 cells/mouse) with 100 uL intratumorally as follows: Group 1-Saline Control (n=7), Group 2-SYN1704 (n=7), Animals were dosed bi-weekly (BIW) according to their grouping either with saline or with the strains intratumorally (IT). Animals were weighed and the tumor volume measured twice weekly. Animals were euthanized when the tumors reached ~2000mm . Plasma and tumor tissue were harvested and kynurenine and tryptophan concentrations were measured by LC/MS as described herein. Results are shown in FIG. 15. A significant reduction in intratumoral (P<0.001) and plasma (P<0.005) concentration of kynurenine was observed for the kynurenine consuming strain SYN1704, while tryptophan levels remained constant.

Example 35. Evaluation of efficacy on indole producing strains on CNS

inflammation in an EAE model

[01241] To assess the effect of administration of various indole producing strains on CNS inflammation, an autoimmune encephalitis mouse model is used. The following strains are used: tryptophan producing strain SYN2339 comprising

AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSOOl), tetR-Ptet-aroGfbr (pl5A); indole-3 acetic acid producing strain SYN2342 comprising (AtrpRAtnaA, tetR-Ptet- trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr-trpDH-ipdC-iadl (pl5A); and tryptamine producing strain SYN2794, comprising AtrpRAtnaA,

(pSClOl),

(pl5A); SYN94 control Nissle.

[01242] To prepare the cells for this study, bacterial growth and induction conditions are as follows. Overnight cultures (5mL) are grown with Strep (control Nissle) or Carbenicillin (engineered strains). 500mL LB flasks are inoculated with the overnight cultures., and grown for 2h at 37C with 250rpm. Next tetracycline (ATC lOOng/mL) is added for 2 hours. Cultures are spun down at 4C for 30min, at 4,000 rpm and the pellets are resuspended in lOmL formulation buffer (PBS, 15% glycerol, 2g/L glucose, 3mM thymidine), aliquoted in 2 ml cryovials and kept at -80C.

[01243] Mice are obtained from Charles River Labs. EAE is induced in eight to ten weeks old mice by subcutaneous immunization with 200 μg MOG35-55 peptide emulsified in complete Freund' s adjuvant (CFA, Difco Laboratories) per mouse, followed by administration of 200 ng pertussis toxin (PTX, List biological laboratories, Inc.) on days 0 and 2 as described (see e.g., Rothhammer et al., Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor; Nat Med. 2016 Jun; 22(6): 586-597, the contents of which is herein incorporated by reference in its entirety). Clinical signs of EAE are assessed according to the following score: 0, no signs of disease; 1, loss of tone in the tail; 2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; 5, moribund.

[01244] Starting from day 22, after EAE induction (day 1 of the study) animals are randomized into treatment groups. Mice are bled and and urine is collected (T=0) to obtain baseline plasma and urine tryptophan and indole levels. Mice are grouped as follows: Group 1: H20 (n=10); Group 2: wild type Nissle with streptomycin resistance (n=10); Group 3: SYN2339 strain (n=10); Group 4: SYN2342 strain (n=10); Group 5: SYN2794 strain (n=10); Group 6: indole, indole-3-propionic acid, indole-3- aldehyde at 400 μg/20g BW For Groups 1 and 3, mice are gavaged with le9 CFUs/dose in 250 ul/dose in the am and pm (2 doses per day), and ATC (20 ng/mL) and 5% sucrose is added to the drinking water. Group 1 is dosed with 250 ul H20. Group 6 is treated daily with indole-3 propionic acid at 400 μg/20g BW via oral gavage (positive control).

[01245] On day 2, animals are dosed twice daily with 250 ul bacteria

(lOelO CFU/dose) or H20 (Group 1) or once daily with indole-3 propionic acid (Group 6). On day 3, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or water or one daily with indole-3 propionic acid (Group 6), and animals are weighed, scored for EAE, blood is drawn and collected and urine is collected at 1 hour post last dose and stored on ice for LC/MS analysis. On days 4-6, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or H20 (Group 1) or once daily with indole-3 propionic acid (Group 6). On day 7, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or water or one daily with indole-3 propionic acid (Group 6), and animals are weighed, scored for EAE, blood is drawn and collected and urine is collected at 1 hour post last dose and stored on ice for LC/MS analysis. On day 8-13, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or H20 (Group 1) or once daily with indole-3 propionic acid (Group 6). On day 14, animals are dosed twice daily with 250 ul bacteria (lOelO CFU/dose) or water or one daily with indole-3 propionic acid (Group 6), and animals are weighed, scored for EAE, blood is drawn and collected and urine is collected at 1 hour post last dose and stored on ice for LC/MS analysis. Animals are sacrificed and astrocytes are isolated and assessed for Ccl2 and Nos2 levels, as described in Rothhammer et al. Blood and urine are analyzed for tryptophan and indole derivatives.

[01246] In a second study, the effects of administration of the tryptophan and/or indole metabolite producing strains is tested in after treatment with antibiotics (Ampicillin 6 mg/20 g body weight (BW), vancomycin 3 mg/20 g BW) to test whether the strains can overcome the ampicillin-mediated interference with disease recovery.

[01247] In other studies, a Ahr knock out mouse model, e.g., a mouse model with specific knockout of Ahr in astrocytes (as described in Rothhammer et al.),is used to assess the role of Ahr in mechanistic studies.

Example 36. Generation of an Auxotroph, e.g., AThyA

[0219] An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.

[0220] A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a lOOum concentration are found in Table 63.

Table 63. Primer Sequences

[0221] For the first PCR round, 4x50ul PCR reactions containing lng pKD3 as template, 25ul 2xphusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions: step 1: 98c for 30s step 2: 98c for 10s step 3: 55c for 15s step 4: 72c for 20s repeat step 2-4 for 30 cycles step 5: 72c for 5min [0222] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30ul nuclease free water.

[0223] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[0224] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2.

Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by fit sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. (thyA auxotrophs will only grow in media

supplemented with thy 3mM).

[0225] Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. ImL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in ImL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for lhours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and lOOug/ml carbenicillin and grown at 30°C for 16-24 hours. Next, transformants were colony purified no n- selectively (no antibiotics) at 42°C.

[0226] To test the colony-purified transformants, a colony was picked from the

42°C plate with a pipette tip and resuspended in ΙΟμί LB.

of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re- streaked on an LB plate to get single colonies, and grown overnight at 37°C.

Example 37. Nissle residence

[0227] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non- limiting example using a streptomycin-resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.

[0228] C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation (i.e., day 0), streptomycin-resistant Nissle (SYN-UCD103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 63. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

Table 63: CFU administered via oral gavage CFU administered via oral gavage

Strain Day 1 Day 2 Day 3

SYN-UCD103 1 .30E+08 8.50E+08 1 .90E+09

[0229] On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs.

1-6; Table 13). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

[0230] Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 64.

[0231] FIG. 44 depicts a graph of Nissle residence in vivo. Streptomycin- resistant Nissle was administered to mice via oral gavage without antibiotic pre- treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse

gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

Table 64. Nissle residence in vivo

2 6.00E+03 7.00E+02 6.00E+02 O.OOE+00 O.OOE+00

3 1.00E+02 2.00E+02 O.OOE+00 O.OOE+00 O.OOE+00

4 1.50E+03 1.00E+02 O.OOE+00 O.OOE+00

5 3.10E+04 3.60E+03 O.OOE+00 O.OOE+00

6 1.50E+03 1.40E+03 4.20E+03 1.00E+02 O.OOE+00

Avg 8.20E+03 1.28E+03 2.28E+03 1.08E+03 4.62E+02

Example 38. Intestinal Residence and Survival of Bacterial Strains in vivo

[0232] Localization and intestinal residence time of streptomycin resistant

Nissle, FIG. 45. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.

[0233] Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 1010 CFU/mL. Mice (C57BL6/J, 10-12 weeks old) were gavaged with 100 μΐ_^ of bacteria (approximately 109 CFU). Drinking water for the mice was changed to contain 0.1 mg/mL

anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post-gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.

[0234] In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37°C overnight, and colonies were counted. The amount of bacteria and residence time of streptomycin resistant Nissle seen in each compartment is shown in FIG. 45.

Example 39. FNR promoter activity

[0235] In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 4A and 4B. The nucleotide sequences of these constructs are shown in Table 65 through Table 69. However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity, exemplary results are shown in FIG. 82.

[0236] Table 65 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Ρ¾τΐ· The construct comprises a translational fusion of the Nissle nirBl gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^th codon of the lacZ coding region. The Pfnri sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0237] Table 66 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf_m2. The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^th codon of the lacZ coding region. The Pfnr2 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0238] Table 67 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P¾r₃. The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The Pf_m3 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0239] Table 68 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf_m4. The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The Pf_nr4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0240] Table 69 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS. The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrSl, fused to lacZ. The P_fms sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 65. Pfnrl-lacZ construct Sequences

Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 200)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtcgtccggcc ttttcctctcttactctgctacgtacatctatttctataaatccgttcaatttgtctgttttttgcacaa acatgaaatatcagacaattccgtgacttaagaaaatttatacaaatcagcaatataccccttaaggagt atataaaggtgaatttgatttacatcaataagcggggttgctgaatcgttaaggtaggcggtaatagaaa agaaatcgaggcaaaaATGagcaaagtcagactcgcaattat GGATC CTCTGGCCGTCGTATTACAACGT CGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGC GTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTT TGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACT GTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCA TTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATATTGA TGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGG TGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTT TACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCA GGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGC GAT T T C C AAGT T AC C AC T C T C T T TAAT GAT GAT T T C AGC C GC GC GGT AC T GGAGGC AGAAGT T C AGAT GT ACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGG CACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTG AACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACA CCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGG TCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCAT GGT CAGGT CAT GGAT GAGCAGAC GAT GGT GC AGGAT AT C C T GC T GAT GAAGC AGAAC AAC T T T AAC GC C G TGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGT GGAT GAAGC CAAT AT T GAAAC C C AC GGC AT GGT GC C AAT GAAT C GT C T GAC C GAT GAT CCGCGCTGGCTA CCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGT C GC T GGGGAAT GAAT CAGGC CAC GGC GC TAAT CAC GAC GCGCTGTATCGCT GGAT CAAATCTGTC GAT CC TTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTAC GCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGC CTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGC TAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAG TCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGA ACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGC AAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTG TTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTG AAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAG CGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACAC ATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCA Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 200)

TCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCG CCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAG TTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGG TCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACT TGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGG AAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACAC CGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCT GGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCA GACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATG GCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAAC CAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATT GGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACC AGTTGGTCTGGTGTCAAAAATAA

Table 66. Pfnr2-lacZ construct sequences

Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 201)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatgcatgcatc aaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacaggagtatttatattgcgcccgtt acgtgggcttcgactgtaaatcagaaaggagaaaacacctATGacgacctacgatcgGGATCCTCTGGCC GTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCC CTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAA TGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTT CCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCA ACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCT CACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCG GCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTG ACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAG TTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCG ACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGG CAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAAC GCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGC GTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAG TGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGT GCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAG CATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGA ACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTA CGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGAT GATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGA GTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGAT CAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATT ATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAA AATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAG TCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGG GACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATT TTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCC GGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTG ACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGC CGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACT GCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGG TCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCT CCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCG TTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACC CCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTG ACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTG CACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACC Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 201)

TTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGG TGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGT AAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGG GATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGC GCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACA ACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGT TTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCG GTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 67. Pfnr3-lacZ construct Sequences

Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 202)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtcgtccggcc ttttcctctcttactctgctacgtacatctatttctataaatccgttcaatttgtctgttttttgcacaa acatgaaatatcagacaattccgtgacttaagaaaatttatacaaatcagcaatataccccttaaggagt atataaaggtgaatttgatttacatcaataagcggggttgctgaatcgttaaGGATCCctctagaaataa ttttgtttaactttaagaaggagatatacatATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAA CGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCT GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCG CTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGAT ACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATC CCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATAT TGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTG TGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCAT TTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGA TCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATC AGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGA TGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAG CGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGC CTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGC ACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAA TGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTG CATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACG CCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGT GGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGG CTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCT GGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGA TCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATG TACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGC TGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTT CGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGAT CAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGC CGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGA AGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATAC CTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCG GTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGA GAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGA CACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACG CCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAA CCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGAT CAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCT GGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATAC ACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGC CGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATA Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 202)

CACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGG CCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTG TCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATT ATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGA AACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGG ATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATT ACCAGTTGGTCTGGTGTCAAAAATAA

Table 68. Pfnr4-lacZ construct Sequences

Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 203)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatgcatgcatc aaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacaggagtatttatattgcgcccGGA

TCCctctagaaataattttgtttaactttaagaaggagatatacatATGACTATGATTACGGATTCTCTG

GCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATC CCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT GAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGAT CTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACA CCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTC GCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAAC TCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAAT TTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGG CAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAA CCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGG AGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGA AACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGAT CGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTG CAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGA GGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCAC GAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGC AGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCG CTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACC GATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACC CGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTG GATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGAT ATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCA AAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAA CAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTC TGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTG ATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCA TCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAA GTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCA AGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGA ACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCA TGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCC CCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAA GCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTG ACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCA TTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCA GTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAA ACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTG CGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCG GGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGC TGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGA Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 203)

CGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCA ACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGAC GGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCG CCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 69. Pfnrs-lacZ construct Sequences

Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 204)

GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgtaacaaaagcaa tttttccggctgtctgtatacaaaaacgccgtaaagtttgagcgaagtcaataaactctctacccattca gggcaatatctctcttGGATCCctctagaaataattttgtttaactttaagaaggagatatacatATGCT

ATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTA ATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTC CCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAA AGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTT ACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAA TCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATT ATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACA GCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGT GCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGAC GTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATT TCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGT TTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAG CGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAA TCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTG CGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATT CGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGG ATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTG GTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTG CCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGC AGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCA CGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCC GACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGG TGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATA TGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGT TTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGT GGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTT TGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTA TCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGA TGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCA GTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTG CAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAA ACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTG CATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGC GATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCG TAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGC CGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCG TGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGG TCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCT GGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACT GCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAA ACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAA CATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGC ACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGG Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 204)

CGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Example 40. Nitric oxide-inducible reporter constructs

[0241] ATC and nitric oxide-inducible reporter constructs were synthesized

(Genewiz, Cambridge, MA). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations (Fig. 42); promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide- inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are |boxed|

Table 70. SEQ ID NO: 205

SEQ ID NO: 205 ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgt tgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgcaccg ccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgggcccat acactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgtt cgggcggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccg cctttgaccagcgcggtaaccactttcatcaaatggcttttggaaatgccgtaggtcga ggcgatggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgc

aattttaaactctagaaataattttgtttaactttaagaaggagatatacat tggcta gcaaaggcgaagaattgttcacgggcgttgttcctattttggttgaattggatggcgat gttaatggccataaattcagcgttagcggcgaaggcgaaggcgatgctacgtatggcaa attgacgttgaaattcatttgtacgacgggcaaattgcctgttccttggcctacgttgg ttacgacgttcagctatggcgttcaatgtttcagccgttatcctgatcatatgaaacgt catgatttcttcaaaagcgctatgcctgaaggctatgttcaagaacgtacgattagctt caaagatgatggcaattataaaacgcgtgctgaagttaaattcgaaggcgatacgttgg ttaatcgtattgaattgaaaggcattgatttcaaagaagatggcaatattttgggccat aaattggaatataattataatagccataatgtttatattacggctgataaacaaaaaaa tggcattaaagctaatttcaaaattcgtcataatattgaagatggcagcgttcaattgg ctgatcattatcaacaaaatacgcctattggcgatggccctgttttgttgcctgataat cattatttgagcacgcaaagcgctttgagcaaagatcctaatgaaaaacgtgatcatat ggttttgttggaattcgttacggctgctggcattacgcatggcatggatgaattgtata aat aat aa

[0242] These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600= -0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half- life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units.

[0243] FIG.42D NO-GFP constructs (the dot blot) E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1 : 100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by

centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR- regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS -treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

[0244] Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR- inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1: 100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. FIG. 42D shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

Claims

Claims

1. A genetically engineered bacterium comprising one or more gene sequences for the modulation of tryptophan and/or tryptophan metabolites in the blood or gut of a mammal.

2. The genetically engineered bacterium of claim 1, wherein tryptophan levels are increased in the blood or gut of a mammal.

3. The genetically engineered bacterium of claim 1, wherein tryptophan levels are decreased in the blood or gut of a mammal.

4. The genetically engineered bacterium of claim 1, wherein kynurenine levels are decreased in the blood or gut of a mammal.

5. The genetically engineered bacterium of claim 1, wherein kynurenine levels are increased in the blood or gut of a mammal.

6. The genetically engineered bacterium of claim 1, wherein tryptamine levels are decreased in the blood or gut of a mammal.

7. The genetically engineered bacterium of claim 1, wherein tryptamine levels are increased in the blood or gut of a mammal.

8. The genetically engineered bacterium of claim 1, wherein indole- 3 -acetic acid levels are decreased in the blood or gut of a mammal.

9. The genetically engineered bacterium of claim 1, wherein indole- 3 -acetic acid levels are increased in the blood or gut of a mammal.

10. The genetically engineered bacterium of claim 1, wherein indole-3-propionic acid levels are decreased in the blood or gut of a mammal.

11. The genetically engineered bacterium of claim 1, wherein indole-3-propionic acid levels are increased in the blood or gut of a mammal.

A genetically engineered bacterium comprising at least one gene or

encoding one or more enzymes for the production of tryptophan.

13. The bacterium of claim 12, comprising gene sequence encoding TrpE.

14. The bacterium of claim 12 comprising gene sequence encoding feedback resistant TrpE.

15. The bacterium of any one of claims 12-14, comprising gene sequence encoding trpDCBA.

16. The bacterium of any one of claims 12-15, comprising gene sequence encoding aroG.

17. The bacterium of any one of claims 12-16, comprising gene sequence encoding feedback resistant aroG (aroGfbr).

18. The bacterium of any one of claims 12-17, comprising gene sequence encoding SerA.

19. The bacterium of any one of claims 12-18, comprising feedback resistant SerA (SerAfbr).

20. The bacterium of any one of claims 12-19, wherein the bacterium comprises an endogenous TnaA gene which is knocked down via mutation or deletion.

21. The bacterium of any one of claims 12-20, wherein the bacterium comprises an endogenous trpR gene which is knocked down via mutation or deletion.

22. The bacterium of any one of claims 12-21, wherein the tryptophan production activity is increased compared to the wild type bacterium.

23. A genetically engineered bacterium comprising gene sequence for the degradation of kynurenine.

24. The genetically engineered bacterium of claim 23, comprising gene sequence encoding one or more kynureninase polypeptide(s).

25. The genetically engineered bacterium of claim 24, wherein the kynureninase is from Pseudomonas fluorescens.

26. The bacterium of claim 24 or claim 25, wherein the bacterium comprises an endogenous trypE gene which is knocked down via mutation or deletion.

27. The bacterium of any one of claims 24-26, wherein the bacterium comprises an endogenous tyrB gene which is knocked down via mutation or deletion.

28. The bacterium of any one of claims 23-27, further comprising gene sequences encoding one or more enzymes for the production of tryptophan.

29. The bacterium of claim 28, comprising gene sequence encoding trpDCBA.

30. The bacterium of claim 28 or claim 29 comprising an endogenous trpE gene which is knocked down via mutation or deletion.

31. The bacterium of any one of claims 28-30 comprising a feedback resistant trpE gene.

32. The bacterium of any one of claims 28-3 lcomprising an endogenous trpR gene which is knocked down via mutation or deletion.

33. The bacterium of any one of claims 28-32 comprising an endogenous tnaA gene which is knocked down via mutation or deletion.

34. The bacterium of any one of claims 28-33 comprising an endogenous tyrB gene which is knocked down via mutation or deletion.

35. The bacterium of any one of claims 28-34 comprising a feedback resistant aroG gene.

36. The bacterium of any one of claims 28-35 comprising a feedback resistant serA gene.

37. The bacterium of one of claims 28-36, wherein the kyureninase activity is increased compared to the wild type bacterium.

38. A genetically engineered bacterium comprising at least one gene or gene cassette encoding one or more enzymes for the production of tryptamine.

39. The bacterium of claim 38, comprising gene sequence(s) encoding tryptophan decarboxylase (Tdc).

40. The bacterium of claim 38 or claim 39, wherein Tdc is from Clostridium sporogenes.

41. The bacterium of any one of claims 38-39, further comprising one or more gene sequence(s) encoding enzymes for the production of tryptophan.

42. The bacterium of claim 41, comprising gene sequence encoding trpDCBA.

43. The bacterium of claim 41 or claim 42 comprising an endogenous trpE gene which is knocked down via mutation or deletion.

44. The bacterium of any one of claims 41-43 comprising a feedback resistant trpE gene.

45. The bacterium of any one of claims 41-44 comprising an endogenous trpR gene which is knocked down via mutation or deletion.

46. The bacterium of any one of claims 41-45 comprising an endogenous tnaA gene which is knocked down via mutation or deletion.

47. The bacterium of any one of claims 41-46 comprising a feedback resistant aroG gene.

48. The bacterium of any one of claims 41-47 comprising a feedback resistant serA gene.

49. The bacterium of one of claims 38-48, wherein the tryptamine production activity is increased compared to the wild type bacterium.

50. A genetically engineered bacterium comprising at least one gene or gene cassette encoding one or more enzymes for the production of indole- 3 -acetic acid.

51. The bacterium of claim 50, comprising gene sequence encoding tryptophan dehydrogenase (trpDH).

52. The bacterium of claim 50 or claim 51, comprising gene sequence encoding Indole-3-pyruvate decarboxylase (ipdC).

53. The bacterium of any one of claims 50-52, comprising gene sequence encoding Indole- 3 -acetaldehyde dehydrogenase (iadl).

54. The bacterium of any one of claims 50-53, comprising gene sequence encoding enzymes for the production of tryptophan.

55. The bacterium of claim 54, comprising gene sequence encoding trpDCBA.

56. The bacterium of claim 54 or claim 55 comprising an endogenous trpE gene which is knocked down via mutation or deletion.

57. The bacterium of any one of claims 54-56 comprising a feedback resistant trpE gene.

58. The bacterium of any one of claims 54-57 comprising an endogenous trpR gene which is knocked down via mutation or deletion.

59. The bacterium of any one of claims 54-58 comprising an endogenous tnaA gene which is knocked down via mutation or deletion.

60. The bacterium of any one of claims 54-59 comprising a feedback resistant aroG gene.

61. The bacterium of any one of claims 54-60 comprising a feedback resistant serA gene.

62. The bacterium of any one of claims 50-61, wherein the indole- 3 -acetic acid production activity is increased compared to the wild type bacterium.

63. The bacterim of any one of claims 1-62, wherein tryptophan uptake is lowered compared to the wild type bacterium.

64. The bacterium of any one of claims 1-63, wherein the bacterium is a thyA auxotroph.

65. The bacterium of any one of claims 1-63, wherein the bacterium comprises an antibiotic resistance gene.

66. The bacterium of any one of claims 12-65, wherein the gene or gene cassette encoding one or more enzymes for the production of tryptophan is operably linked to a directly or indirectly inducible promoter.

67. The bacterium of claim 66, wherein the promoter is induced by exogenous environmental conditions found in a mammalian gut.

68. The bacterium of claim 67, wherein the promoter is induced under low-oxygen or anaerobic conditions.

69. The bacterium of claim 68, wherein the promoter is an FNR promoter selected from nirB l, nirB2, nirB3, ydfZ, fnrS l, and fnrS2.

70. The bacterium of claim 66, wherein the gene or gene cassette encoding one or more enzymes for the production of tryptophan is operably linked to a constitutive promoter.

71. The bacterium of claim 70, wherein the constitutive promoter is selected from a promoter listed in Tables 10-20.

72. The bacterium of any one of claims 1-71, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.

73. The bacterium of claim 72, wherein the bacterium is Escherichia coli strain Nissle.

74. The bacterium of any one of claims 12-73, comprising one or more gene sequences encoding a gut barrier enhancer molecule and/or an ant i- inflammatory effector.

75. The bacterium of claim 74, wherein the gut barrier enhancer molecule and/or anti- inflammatory effector is selected from a short chain fatty acid, an antiinflammatory cytokine, and Glp-2.

76. The bacterium of claim 75, wherein in the short chain fatty acid is butyrate.

77. The bacterium of claim 75, wherein the anti- inflammatory cytokine is selected from IL-10 and IL-22.

78. The bacterium of claim 75, wherein the gut barrier enhancer molecule and/or anti- inflammatory effector is Glp2.

79. The bacterium of any one of claims 12-22, wherein the bacterium further comprises gene sequences encoding a checkpoint inhibitor and/or a pro-inflammatory cytokine.

80. The bacterium of claim 79, wherein the immune checkpoint inhibitor is selected from anti-PD-1, anti-PD-Ll, anti-LAG3, anti-TIMl, anti-CTLA4 antibodies.

81. The bacterium of claim 80, wherein the immune checkpoint inhibitor is an anti- PD-1 antibody.

82. The bacterium of claim 80, wherein the immune checkpoint inhibitor is an anti- PD-Ll antbody.

83. The bacterium of 79, wherein the pro-inflammatory cytokine is IL-15.

84. The bacterium of any one of claims 12-22, wherein the bacterium is coadministered with a checkpoint inhibitor.

85. The bacterium of claim 84, wherein the checkpoint inhibitor is PD-1.

86. A pharmaceutically acceptable composition comprising the genetically engineered bacterium of any one of claims 1-85 and a pharmaceutically acceptable carrier.