CA3013770A1 - Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier - Google Patents

Abstract

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of treating or preventing autoimmune disorders, inhibiting inflammatory mechanisms in the gut, and/or tightening gut mucosal barrier function are disclosed.

Description

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2 Bacteria Engineered to Treat Diseases that Benefit from Reduced Gut Inflammation and/or Tightened Gut Mucosal Barrier RELATED APPLICATIONS
[01] This application is a continuation-in-part of PCT Application No.
PCT/US2016/020530, filed March 2, 2016; PCT Application No. PCT/US2016/050836, filed September 8, 2016, and U.S. Application No. 15/260,319, filed September 8, 2016;
and claims the benefit of U.S. Provisional Application No. 62/291,461 filed February 4, 2016; U.S. Provisional Application No. 62/291,468 filed February 4, 2016; U.S.

Provisional Application No. 62/291,470 filed February 4, 2016; U.S.
Provisional Application No. 62/347,508, filed June 8, 2016; U.S. Provisional Application No.
62/354,682, filed June 24, 2016; U.S. Provisional Application No. 62/362,954, filed July 15, 2016; U.S. Provisional Application No. 62/385,235, filed September 8, 2016; U.S.
Provisional Application No. 62/423,170, filed November 16, 2016; U.S.
Provisional Application No. 62/439,871, filed December 28, 2016; PCT Application No.
PCT/U52016/032565, filed May 13, 2016; U.S. Provisional Application No.
62/347,576, filed June 8, 2016; U.S. Provisional Application No. 62/348,620, filed June 10, 2016; PCT
Application No. PCT/U52016/039444, filed June 24, 2016; and PCT Application No.
PCT/U52016/069052, filed December 28, 2016. The entire contents of each of the foregoing applications are expressly incorporated herein by reference in their entireties to provide continuity of disclosure.
BACKGROUND OF THE INVENTION
[02] This disclosure relates to compositions and therapeutic methods for inhibiting inflammatory mechanisms in the gut, restoring and tightening gut mucosal barrier function, and/or treating and preventing autoimmune disorders. In certain aspects, the disclosure relates to genetically engineered bacteria that are capable of reducing inflammation in the gut and/or enhancing gut barrier function. In some embodiments, the genetically engineered bacteria are capable of reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing an autoimmune disorder. In some aspects, the compositions and methods disclosed herein may be used for treating or preventing autoimmune disorders as well as diseases and conditions associated with gut inflammation and/or compromised gut barrier function, e.g., diarrheal diseases, inflammatory bowel diseases, and related diseases.

[03] Inflammatory bowel diseases (IBDs) are a group of diseases characterized by significant local inflammation in the gastrointestinal tract typically 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.
Current approaches to treat IBD are focused on therapeutics that modulate the immune system and suppress inflammation. These therapies include steroids, such as prednisone, and tumor necrosis factor (TNF) inhibitors, such as Humira (Cohen et al., 2014).
Drawbacks from this approach are associated with systemic immunosuppression, which includes greater susceptibility to infectious disease and cancer.

[04] Other approaches have focused on treating compromised barrier function by supplying the short-chain fatty acid butyrate via enemas. Recently, several groups have demonstrated the importance of short-chain fatty acid production by commensal bacteria in regulating the immune system in the gut (Smith et al., 2013), showing that butyrate plays a direct role in inducing the differentiation of regulatory T cells and suppressing immune responses associated with inflammation in IBD (Atarashi et al., 2011;
Furusawa et al., 2013). Butyrate is normally produced by microbial fermentation of dietary fiber and plays a central role in maintaining colonic epithelial cell homeostasis and barrier function (Hamer et al., 2008). Studies with butyrate enemas have shown some benefit to patients, but this treatment is not practical for long term therapy. More recently, patients with IBD
have been treated with fecal transfer from healthy patients with some success (Ianiro et al., 2014). This success illustrates the central role that gut microbes play in disease pathology and suggests that certain microbial functions are associated with ameliorating the IBD
disease process. However, this approach raises safety concerns over the transmission of infectious disease from the donor to the recipient. Moreover, the nature of this treatment has a negative stigma and thus is unlikely to be widely accepted.

[05] Compromised gut barrier function also plays a central role in autoimmune diseases 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 and both intestinal and extraintestinal autoimmune disorders can occur" (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). Rheumatoid arthritis and celiac disease, for example, are autoimmune disorders that are thought to involve increased intestinal permeability (Lerner et al., 2015b). 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).

[06] 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). Others have shown that gut bacteria are a potential therapeutic target in the prevention of asthma and exhibit strong immunomodulatory capacity... in lung inflammation (Arrieta 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.

[07] Recently there has been an effort to engineer microbes that produce anti-inflammatory molecules, such as IL-10, and administer them orally to a patient in order to deliver the therapeutic directly to the site of inflammation in the gut. The advantage of this approach is that it avoids systemic administration of immunosuppressive drugs and delivers the therapeutic directly to the gastrointestinal tract. However, while these engineered microbes have shown efficacy in some pre-clinical models, efficacy in patients has not been observed. One reason for the lack of success in treating patients is that the viability and stability of the microbes are compromised due to the constitutive production of large amounts of non-native proteins, e.g., human interleukin. Thus, there remains a great need for additional therapies to reduce gut inflammation, enhance gut barrier function, and/or treat autoimmune disorders, and that avoid undesirable side effects.
Summary

[08] The genetically engineered bacteria disclosed herein are capable of producing therapeutic anti-inflammation and/or gut barrier enhancer molecules.
In some embodiments, the genetically engineered bacteria are functionally silent until they reach an inducing environment, e.g., a mammalian gut, wherein expression of the therapeutic molecule is induced. In certain embodiments, the genetically engineered bacteria are naturally non-pathogenic and may be introduced into the gut in order to reduce gut inflammation and/or enhance gut barrier function and may thereby further ameliorate or prevent an autoimmune disorder. In certain embodiments, the anti-inflammation and/or gut barrier enhancer molecule is stably produced by the genetically engineered bacteria, and/or the genetically engineered bacteria are stably maintained in vivo and/or in vitro.
The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of treating diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier function, e.g., an inflammatory bowel disease or an autoimmune disorder.

[09] In some embodiments, the genetically engineered bacteria of the invention produce one or more therapeutic molecule(s) under the control of one or more promoters induced by an environmental condition, e.g., an environmental condition found in the mammalian gut, such as an inflammatory condition or a low oxygen condition. In on-limiting exemplary embodiments, the genetically engineered bacteria produce one or more therapeutic molecule(s) 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. In some embodiments, the therapeutic molecule is butyrate; in an inducing environment, the butyrate biosynthetic gene cassette is activated, and butyrate is produced. Local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria produce their therapeutic effect only in inducing environments such as the gut, thereby lowering the safety issues associated with systemic exposure.

[010] Disclosed herein is a butyrate-producing bacterium comprising at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has at least one mutation in or deletion of an endogenous pta gene. Such bacterium is capable of producing butyrate, but does not produce acetate. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous frd gene.
In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene, an endogenous frd gene, and an endogenous ldhA gene. In certain specific embodiments, the butyrate-producing bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has at least one mutation in or deletion of an endogenous pta gene and at least one mutation in or deletion of an endogenous gene selected from adhE gene and/or ldhA gene and/or frd gene.

[011] In any of the above described embodiments of butyrate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature. In any of the above described embodiments of butyrate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut.

[012] In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous adhE gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous pta gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous gene selected from frd and/or ldhA and/or adhE and/or pta. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous ldhA gene, frd gene, adhE gene, and pta gene.

[013] In some embodiments, the bacterium described above comprises an endogenous pta gene and produces acetate. In these embodiments, the bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has an endogenous pta gene. Such bacterium is capable of producing butyrate and acetate. In some embodiments of this bacterium, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous ldhA
gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene, an endogenous frd gene, and an endogenous ldhA gene. In certain specific embodiments, the butyrate-producing bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA
and wherein the bacterium has an endogenous pta gene and at least one mutation in or deletion of an endogenous gene selected from adhE gene and/or ldhA gene and/or frd gene.

[014] In any of the above-described embodiments of butyrate-producing bacterium, the at least one gene or gene cassette for producing butyrate may comprise ter, thiAl, hbd, crt2, pbt, and buk genes. In any of the above-described embodiments of butyrate-producing bacterium, the at least one gene or gene cassette for producing butyrate may comprise ter, thiAl, hbd, crt2, and tesB genes.

[015] In any of the above described embodiments of butyrate- and acetate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature. In any of the above described embodiments of butyrate-and acetate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut.

[016] In another aspect, disclosed herein is an acetate-producing bacterium that produces acetate but not butyrate. In any of these embodiments, the acetate-producing bacterium produces acetyl CoA and comprises a wild-type pta gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a ldhA gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhE gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a frd gene.
In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an ldhA gene and at least one mutation in or deletion of an adhE gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a ldhA gene and at least one mutation in or deletion of an frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhA gene and at least one mutation in or deletion of an frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhA gene, at least one mutation in or deletion of an frd gene, and at least one mutation in or deletion of an ldhA gene.

[017] The bacterium may produce an increased level of acetate as compared to a bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and has an endogenous frd gene and/or endogenous ldhA gene and/or endogenous adhA gene.
The bacterium may produce an increased level of acetate as compared to a bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and does not comprise at least one mutation in or deletion of an ldhA gene, an adhE gene, and/or a frd gene.

[018] In any of the above-described embodiments comprising a gene or gene cassette for producing butyrate in which the gene or gene cassette is operably linked to a directly or indirectly inducible promoter, the promoter may be induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter. In some embodiments, the promoter is an FNR-responsive promoter. In some embodiments, the promoter may be induced by the presence of reactive nitrogen species. In some embodiments, the promoter is selected from an NsrR-responsive promoter, NorR-responsive promoter, and a DNR-responsive promoter. In some embodiments, the promoter may be induced by the presence of reactive oxygen species. In some embodiments, the promoter is selected from an OxyR-responsive promoter, PerR-responsive promoter, OhrR-responsive promoter, SoxR-responsive promoter, or a RosR-responsive promoter.

[019] In some embodiments, the gene and/or gene cassette is located on a chromosome in the bacterium. In some embodiments, the at least one gene and/or gene cassette is located on a plasmid in the bacterium.

[020] In some embodiments, the bacterium is a probiotic bacterium. In some embodiments, the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus. In some embodiments, thebacterium is Escherichia coli strain Nissle.

[021] In some embodiments, the bacterium is an an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut. The bacterium may be an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.

[022] Disclosed herein is a pharmaceutically acceptable composition comprising one or more of any of the bacterium disclosed herein; and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for oral or rectal administration.

[023] Disclosed herein is a method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, a composition disclosed herein.

[024] Disclosed herein is a method of treating a disease or condition associated with gut inflammation and/or compromised gut barrier function comprising the step of administering to a patient in need thereof, a composition.

[025] The autoimmune disorder may be selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylo sing 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 muco sal pemphigoid, Crohn's disease, Cogan 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, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS
(Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS
syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds 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.

[026] The autoimmune disorder may be selected from the group consisting of type 1 diabetes, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, celiac disease, and ankylosing spondylitis.

[027] The disease or condition may be selected from an inflammatory bowel disease, including Crohn's disease and ulcerative colitis, and a diarrheal disease.
Brief Description of the Figures

[028] FIG. 1A, FIG. 1B, FIG 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G, FIG.
1H, FIG. 11, FIG. 1J, and FIG. 1K depict schematics of E. coli that are genetically engineered to express a propionate biosynthesis cassette (FIG. 1A), a butyrate biosynthesis cassette (FIG. 1B), an acetate biosynthesis cassette (FIG. 1C), a cassette for the expression of GLP-2 (FIG. 1D), a cassette for the expression of human IL-10 (FIG.
1E) or v-IL-22 or hIL-22 (FIG. 1F) under the control of a FNR-responsive promoter. The genetically engineered E. coli depicted in FIG. 1D, FIG. 1E, and FIG. 1F may further comprise a secretion system for secretion of the expressed polypeptide out of the cell.
FIG. 1Gdepicts bacteria overexpressing butyrate (and not expressing acetate) by expressing a butyrate biosynthesis cassette in combination with deletions in adhE and pta (FIG. 1G), FIG. 1H depicts bacteria overexpressing butyrate by expressing a butyrate biosynthesis cassette in combination with deletions in ldhA, FIG. 11 depicts bacteria overexpressing butyrate by expressing a butyrate biosynthesis cassette in combination with deletions in adhE and frdA (FIG. 1I). FIG. 1J depicts bacteria overexpressing acetate by deletion in ldhA. FIG. 1K depicts bacteria overexpressing GLP-2 in combination with a deletion in adhE and pta.

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

[030] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F depict schematics of the gene organization of exemplary bacteria of the disclosure.
FIG. 3A and FIG. 3B depict the gene organization of an exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions. FIG. 3A
depicts relatively low butyrate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (bcd2, e033, e03, thiAl, hbd, crt2, pbt, and buk; white boxes) is expressed.
FIG. 3B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIG. 3C and FIG. 3D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 3C, in the absence of NO, the NsrR transcription factor (circle, "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) is expressed. In FIG. 3D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[031] FIG. 3E and FIG. 3F depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H202. In FIG. 3E, in the absence of H202, the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) is expressed. In FIG.
3F, in the presence of H202, the OxyR transcription factor interacts with H202 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[032] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F depict schematics of the gene organization of exemplary bacteria of the disclosure.
FIG. 4A and FIG. 4B depict the gene organization of another exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions using a different butyrate circuit from that shown in FIG. 3A, FIG 3B, FIG. 3C, FIG.
3D, FIG.
3E, and FIG. 3F. FIG. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter").
Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, and buk; white boxes) is expressed. FIG. 4B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIG. 4C and FIG. 4D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 4C, in the absence of NO, the NsrR transcription factor (circle, "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk) is expressed.
In FIG. 4D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. FIG. 4E and FIG. 4F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H202. In FIG. 4E, in the absence of H202, the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk) is expressed. In FIG.
4F, in the presence of H202, the OxyR transcription factor interacts with H202 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[033] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F depict schematics of the gene organization of exemplary bacteria of the disclosure.
FIG. 5A and FIG. 5B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, and tesB) is expressed. FIG. 5B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIG. 5C and FIG. 5D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 5C, in the absence of NO, the NsrR transcription factor ( "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, tesB) is expressed. In FIG. 5D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. FIG. 5E and FIG. 5F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H202. In FIG. 5E, in the absence of H202, the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, tesB) is expressed. In Figs. 6F, in the presence of H202, the OxyR transcription factor interacts with H202 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.

[034] FIG. 6A and FIG. 6B depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production. FIG.
6A depicts relatively low propionate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the propionate biosynthesis enzymes (pct, lcdA, lcdB, lcdC, e0, acrB, acrC) is expressed. FIG. 6B depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR
dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO or H202 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C-4F, FIG. 5C-5F.

[035] FIG. 7 depicts an exemplary propionate biosynthesis gene cassette.

[036] FIG. 8A, FIG. 8B, and FIG. 8C depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
FIG. 8A depicts relatively low propionate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pc1) is expressed.
FIG. 8B depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. FIG. 8C depicts an exemplary propionate biosynthesis gene cassette. In other embodiments, propionate production is induced by NO or H202 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C-4F, FIG. 5C-5F.

[037] FIG. 9A and FIG. 9B depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production. FIG.
9A depicts relatively low propionate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, 1pd, tesB) is expressed. FIG. 9B
depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR
dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO or H202 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C-4F, FIG. 5C-5F.

[038] FIG. 10A, FIG. 10B, and FIG. 10C depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure.
FIG. 10A depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. FIG. 10B and FIG.
10C depict schematics of the gene organization of another exemplary engineered bacterium of the invention and its induction of propionate production under low-oxygen conditions. FIG. 10B depicts relatively low propionate production under aerobic conditions in which oxygen (02) prevents (indicated by "X") FNR (boxed "FNR") from dimerizing and activating the FNR-responsive promoter ("FNR promoter").
Therefore, none of the propionate biosynthesis enzymes (sbm, ygfD, ygfG, ygfH) is expressed. FIG.
10C depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO
or H202 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C-4F, FIG. 5C-5F.

[039] FIG. 11 depicts a bar graph showing butyrate production of butyrate producing strains of the disclosure. FIG. 11 shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB
gene (a thioesterase that cleaves off the butyrate portion from butyryl CoA).
Overnight cultures of cells were diluted 1:100 in Lb and grown for 1.5 hours until early log phase was reached at which point anhydrous tet was added at a final concentration of 100ng/m1 to induce plasmid expression. After 2 hours induction, cells were washed and resuspended in M9 minimal media containing 0.5% glucose at 0D600=0.5. Samples were removed at indicated times and cells spun down. The supernatant was tested for butyrate production using LC-MS.

[040] FIG. 12 depicts a bar graph showing butyrate production of butyrate producing strains of the disclosure. FIG. 12 shows butyrate production in strains comprising a tet¨butyrate cassette having ter substitution (pLOGIC046) or the tesB
substitution (ptb-buk deletion), demonstrating that the tesB substituted strain has greater butyrate production.

[041] FIG. 13 depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. Strains depicted are comprising a bcd-butyrate cassette, with or without a nuoB deletion, and comprising a ter-butyrate cassette, with or without a nuoB deletion. Strains with deletion are labeled with nuoB. The NuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains. NuoB
is a main protein complex involved in the oxidation of NADH during respiratory growth.
In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.

[042] FIG. 14A, FIG. 14B, FIG.14C, and FIG. 14D depict schematics and graphs showing butyrate or biomarker production of a butyrate producing circuit under the control of an FNR promoter. FIG. 14A depicts a schematic showing a butyrate producing circuit under the control of an FNR promoter. FIG. 14B depicts a bar graph of anaerobic induction of butyrate production. FNR-responsive promoters were fused to butyrate cassettes containing either the bcd or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes. Cells were washed and resuspended in minimal media w/ 0.5% glucose and incubated microaerobically to monitor butyrate production over time. SYN-501 led to significant butyrate production under anaerobic conditions. FIG. 14C depicts SYN-501 in the presence and absence of glucose and oxygen in vitro. SYN-501 comprises pSC101 PydfZ-ter butyrate plasmid; SYN-500 comprises pSC101 PydfZ-bcd butyrate plasmid;
SYN-506 comprises pSC101 nirB-bcd butyrate plasmid. FIG. 14D depict levels of mouse lipocalin 2 (left) and calprotectin (right) quantified by ELISA using the fecal samples in an in vivo model. SYN-501 reduces inflammation and/or protects gut barrier function as compared to wild type Nissle control.

[043] FIG. 15 depicts a graph measuring gut-barrier function in dextran sodium sulfate (DSS)-induced mouse models of IBD. The amount of FITC dextran found in the plasma of mice administered different concentrations of DSS was measured as an indicator of gut barrier function.

[044] FIG. 16 depicts serum levels of FITC-dextran analyzed by spectrophotometry. FITC-dextran is a readout for gut barrier function in the DSS-induced mouse model of IBD.

[045] FIG. 17 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H20, 100 mM butyrate in H20, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid.
Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only.
Levels are close to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM
butyrate.

[046] FIG. 18 depicts a bar graph comparing butyrate concentrations produced in vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia butyricum MIYARISAN (a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain) under aerobic and anaerobic conditions at the indicated timepoints. The Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.

[047] FIG. 19A depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid copies. Integrated butyrate strains, SYN1001 and SYN1002 (both integrated at the agaI/rsml locus) gave comparable butyrate production to the plasmid strain SYN501.

[048] FIG. 19B and FIG. 19C depict bar graphs showing the effect of the supernatants from the engineered butyrate-producing strain, SYN1001, on alkaline phosphatase activity in HT-29 cells represented in bar (FIG. 19B) and nonlinear fit (FIG.
19C) graphical formats.

[049] FIG. 20A and FIG. 20B depicts the construction and gene organization of an exemplary plasmids. FIG. 20A depicts the construction and gene organization of an exemplary plasmids comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct). FIG.

depicts the construction and gene organization of another exemplary plasmid comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic046- nsrR-norB-butyrogenic gene cassette).

[050] FIG. 21 depicts butyrate production using SYN001 + tet (control wild-type Nissle comprising no plasmid), SYN067 + tet (Nissle comprising the pLOGIC031 ATC-inducible butyrate plasmid), and SYN080 + tet (Nissle comprising the pLOGIC046 ATC-inducible butyrate plasmid).

[051] FIG. 22 depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct (SYN133) or the pLogic046-nsrR-norB-butyrate construct (SYN145), which produce more butyrate as compared to wild-type Nissle (SYN001).

[052] FIG. 23 depicts the construction and gene organization of an exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette (pLogic031-oxyS-butyrogenic gene cassette).

[053] FIG. 24 depicts the construction and gene organization of another exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette (pLogic046-oxyS- butyrogenic gene cassette).

[054] FIG. 25 depicts a schematic illustrating a strategy for increasing butyrate and acetate production in engineered bacteria. Aerobic metabolism through the citric acid cycle (TCA cycle) (crossed out) is inactive in the anaerobic environment of the colon. E.
coli makes high levels of acetate as an end production of fermentation. To improve acetate production, while still maintaining highlevels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of competing routes (shown in in rounded boxes) are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions of interest therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.

[055] FIG. 26A and FIG. 26B depict line graphs showing acetate production over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) (FIG.
26A) and in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 26B). Acetate production of an engineered E. coli Nissle strain comprising a deletion in the endenous ldh gene (SYN2001) was compared with streptomycin resistant Nissle (5YN94).

[056] FIG. 26C and FIG. 26D depict bar graphs showing acetate and butyrate production in 0.5% glucose MOPS (pH6.8) (FIG. 26C) and acetate and butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 26D). Deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes. SYN2006 comprises a FNRS ter-tesB cassette integrated at the HA1/2 locus and a deletion in the endogenous adhE gene. SYN2007 comprises a FNRS ter-tesB
cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene.

comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the endogenous adhE
gene. 5YN2003 comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the endogenous ldhA gene.

[057] FIG. 26E depicts a bar graph showing acetate and butyrate production at the indicated time points post induction in 0.5% glucose MOPS (pH6.8). A
strain comprising a FNRS-ter-tesB butyrate cassette integrated at the HA1/2 locus of the chromosome (SYN1004) was compared with a strain comprising the same integrated cassette and additionally a deletion in the endogenous frd gene (5YN2005).

[058] FIG. 26F depicts a bar graph showing acetate and butyrate production at hours in 0.5% glucose MOPS (pH6.8), comparing three strains engineered to produce short chain fatty acids. SYN2001 comprises a deletion in the endenous ldh gene;
5YN2002 comprises a FNRS-ter-tesB butyrate cassette integrated at the HA1/2 locus and deletions in the endogenous adhE and pta genes. 5YN2003 comprises FNRS-ter-pbt-buk butyrate cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA
gene.

[059] FIG. 26G and FIG. 26H depict line graphs showing the effect of supernatants from the engineered acetate-producing strain, SYN2001, on LPS-induced IFN7 secretion in primary human PBMC cells from donor 1 (D1) (Fig. 26G) and donor 2 (D2) (FIG. 26H).

[060] FIG. 27 depicts a schematic of an exemplary propionate biosynthesis gene cassette.

[061] FIG. 28 depicts a schematic of a construct comprising the sleeping beauty mutase operon from E. coli under the control of a heterologous FnrS promoter.

[062] FIG. 29 depicts a bar graph of proprionate concentrations produced in vitro by the wild type E coli BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in FIG. 28.

[063] FIG. 30A, FIG. 30B, and FIG. 30C 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, GLP-2, IL-10, and IL-22, is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD
promoter (FIG. 30A and FIG. 30B) or a tet-inducible promoter (FIG. 30C). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD).
Optionally, an N
terminal part of FliC is included in the construct, as shown in FIG. 30B and FIG. 30D.

[064] FIG. 31A and FIG. 31B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is 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 1pp, pal, tolA, and/or nlpI.
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.
31A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, FIG. 31B), 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.

[065] FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, and FIG. 32E depict schematics of non-limiting examples of constructs for the expression of GLP2 for bacterial secretion. FIG. 32A depicts a schematic of a human GLP2 construct inserted into the FliC
locus, under the control of the native FliC promoter. FIG. 32B depicts a schematic of a human GLP2 construct, including the N terminal 20 amino acids of FliC, inserted into the FliC locus under the control of the native FliC promoter. FIG. 32C depicts a schematic of a human GLP2 construct, including the N-terminal 20 amino acids of FliC, inserted into the FliC locus under the control of a tet inducible promoter. FIG. 32D depicts a schematic of a human GLP2 construct with a N terminal OmpF secretion tag (sec-dependent secretion system) under the control of a tet inducible promoter. FIG. 32E
depicts a schematic of a human GLP2 construct with a N terminal TorA secretion tag (tat secretion system) under the control of a tet inducible promoter.

[066] FIG. 33A and FIG. 33B depict line graphs of ELISA results. FIG. 33A
depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on extracts from serum-starved Co10205 cells treated with supernatants from engineered bacteria comprising a PAL deletion and an integrated construct encoding hIL-22 with a phoA
secretion tag. The data demonstrate that hIL-22 secreted from the engineered bacteria is functionally active. FIG. 33B depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA showing a antibody completion assay. Extracts from Co10205 cells were treated with the bacterial supernatants from the IL-22 overexpres sing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody. The data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821.

[067] FIG. 33C depicts a line graph showing SYN3001 (PhoA-IL-22 in pal mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3 activation.

[068] FIG. 33D depicts a line graph showing that anti IL-22 neutralizing antibody inhibits SYN3001-induced STAT3 activation (n=3).

[069] FIG. 33E depicts a Western blot analysis of bacterial supernatants from strain SYN2980 and SYN2982, using IL-10 antibody (IL-10 (D13A11) XP Rabbit mAb #12163, Cell Signaling Technology). The secreted polypepetide has the same molecular weight as the standards, indicating that the signal sequence is cleaved from the native peptide.

[070] FIG. 34 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-0-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.

[071] FIG. 35 depicts a schematic of bacterial tryptophan catabolism machinery, which is genetically and functionally homologous to IDO1 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. 35. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG.

35, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde.

[072] FIG. 36A and FIG. 36B depict schematics of indole metabolite mode of action (FIG.36A) and indole biosynthesis (FIG. 36B). FIG.36A depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) 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). FIG. 36B 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 FIGs. 36A and 36B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIGs. 36A and 36B, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde.

[073] FIG. 37A and FIG. 37B depict diagrams of bacterial tryptophan metabolism pathways. FIG. 37A 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 (EC1.1.1.110); 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. 37B 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; IA0x: 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, Cc1b2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125 (CYP79B2, CYP79B3); 9.
EC
1.4.3.2 (Sta0), 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 FIGs. 37A and 37B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIGs. 37A and 37B. 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.

[074] FIG. 38 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.

[075] FIG. 39 depicts one embodiment of the disclosure in which the E. coli TRP
synthesis enzymes are expressed from a construct under the control of a tetracycline inducible system.

[076] FIG. 40A, FIG. 40B, FIG. 40C, and FIG. 40D 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.
In certain embodiments the one or more cassettes are under the control of constitutive promoters. 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 (A thyA; thymidine dependence). FIG. 40A 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. 40B, and/or FIG. 40C, and/or FIG. 40D. FIG. 40B
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. 40A and/or described in the description of FIG. 40A.
The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40C, and/or FIG. 40D. Optionally, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. FIG. 40C 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 NADI 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. 40A and/or described in the description of FIG. 40A.
The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40B, and/or FIG. 40D. 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. 40D 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.
40A and/or described in the description of FIG. 40A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40B, and/or FIG.
40C. 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.

[077] FIG. 41A, FIG. 41B, FIG. 41D, FIG. 41D, FIG. 41E, FIG. 41F, FIG.
41G, and FIG. 41H 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. 41A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. In certain embodiments the one or more cassettes are under the control of inducible promoters. In certain embodiments the one or more cassettes are under the control of constitutive promoters. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG.
40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D 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.
41B 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. 40A
and/or FIG.
40B, and/or FIG. 40C, and/or FIG. 40D 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 sta0 (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-acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 41C 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. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D
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 and/or 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. 41D 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. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D
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. 41E 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.
40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D 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 (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. 41F 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. 40A
and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D 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 (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 (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. 41G 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.
40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D 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. 41H 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. 41A, FIG. 41B, FIG. 41D, FIG. 41D, FIG. 41E, FIG. 41F, FIG. 41G and FIG.
41H 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.

[078] FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In certain embodiments, the one or more cassettes are under the control of inducible promoters. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. In FIG. 42A, the optional circuits for tryptophan production are as depicted and described in FIG. 40A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D. 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 sta0 (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 AA01 (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. 42B the optional circuits for tryptophan production are as depicted and described in FIG. 40A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C
and/or FIG. 40D. 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 and/or Clostridium sporogenes) ot tynA (Monoamine oxidase, e.g., from E. coli) and or iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AA01 (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. 42C the optional circuits for tryptophan production are as depicted and described in FIG. 40A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C
and/or FIG. 40D. 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 sta0 (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.
42D the optional circuits for tryptophan production are as depicted and described in FIG.
40A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D. 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. 42E the optional circuits for tryptophan production are as depicted and described in FIG. 40A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 40B
and/or FIG. 40C and/or FIG. 40D. 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 cyp71a13 (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. 42A, FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E 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.

[079] In FIG. 42F the optional circuits for tryptophan production are as depicted and described in FIG. 40A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D. Alternatively, optionally, tryptophan can be imported through a transporter. 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 (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.

[080] FIG. 43A, FIG. 43B, and FIG. 43C 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. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. 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 (A thyA;
thymidine dependence). FIG. 43A 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. 40A and/or FIG. 40B
and/or FIG.
40C and/or FIG. 40D. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine. FIG. 43B 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. 40A
and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. 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 (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate. FIG. 43C 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. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D.
Additionally, the strain comprises a circuit as described in FIG. 48, comprising trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-3y1)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 indo1-3-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 AcuI: (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 comprisefldH/ and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indo1-3-yl)pyruvate into indole-3-lactate).

[081] FIG. 44A and FIG. 44B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production. FIG.
44A 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 tryptophan(AroH). AroB: 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 cyclization of 3-deoxy-D-arabino-heptulosonic 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-Phosphoshikimate-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 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 (anthranilate 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 carboxyphenylaminodeoxyribulo se 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 (a2-02) tryptophan synthase complex. The TrpB
polypeptide functions as the 0 subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the 0 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. 44B 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. TrpEtbr, and/or TrpD, and/or TrpC, and/or TrpA, 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, two or more of the strategies depicted in the schematic of FIG. 44B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.

[082] FIG.45A and FIG. 45B and FIG. 45C depict bar graphs showing tryptophan production by various engineered bacterial strains. FIG.45A depicts a bar graph showing tryptophan production by various tryptophan producing strains.
The data show expressing a feedback resistant form of AroG (Arodbl.) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE (trpE
tbr) has a positive effect on tryptophan production. FIG. 45B shows tryptophan production from a strain comprising a tet-trpEtbrDCBA, tet-aroGthr 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.
45C depicts a bar graph showing improved tryptophan production by engineered strain comprising AtrpRAtnaA, tet-trperDCBA, tet-arodbr through the addition of serine.

[083] FIG. 46 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-aroGthr-serA, tet-trpEfbrDCBA. SYN2476 comprises AtrpRAtnaA, tet-trpEtbrDCBA. 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.

[084] FIG. 47 depicts a schematic of an indole-3-propionic acid (IPA) 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 Bardrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). In some embodiments, IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole-3-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. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR
and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

[085] FIG. 48 depicts a schematic of indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA) circuits. Enzymes are as follows :
1. TrpDH:
tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108;
FldHl/F1dH2: 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; AcuI:
acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; lad 1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.

[086] Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indo1-3-yl)pyruvate (IPyA), NH3, NAD(P)H and H. Indole-3-lactate dehydrogenase ((EC
1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indo1-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-propionyl-CoA:indole-3-lactate CoA transferase (F1dA ) converts indole-3-lactate (ILA) and indo1-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA.
Indole-3-acrylyl-CoA reductase (F1dD ) and acrylyl-CoA reductase (AcuI) 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. Indole-3-pyruvate decarboxylase (1pdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d) ladl:
Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG.

44 and as described elsewhere herein. In some embodiments, AroG and/or TrpE
are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA
gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.

[087] FIG. 49 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 (pSC101), tetR-Ptet-aroGfbr (p15A)).

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 (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iadl (p15A)). SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 converts all trypophan it produces into IAA.

[088] FIG. 50 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-Ptet-trpEtbrDCBA (pSC101), tetR-PteraroGthr (p15A). SYN2340 comprises AtrpRAtnaA, tetR-Ptet-trpEtbrDCBA (pSC101), tetR-PteraroGtk-tdcc, (p15A). SYN2794 comprises AtrpRAtnaA, tetR-Ptet-trpEtbrDCBA (pSC101), tetR-PteraroGthr-tdccs (p15A).
Results indicate that Tdccs from Clostridium sporo genes is more efficient the Tdcc, from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine.

[089] FIG. 51A, FIG. 51B, FIG. 51C, FIG. 51D, FIG. 51E depict schematics of non-limiting examples of genetically engineered bacteria of the disclosure which comprises one or more gene sequence(s) and/or gene cassette(s) as described herein.

[090] FIG. 52 depicts a map of integration sites within the E. coil 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.

[091] FIG. 53 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).

[092] FIG. 54A and FIG. 54B depict schematics of bacterial chromosomes, for example the E. coli Nissle 1917 Chromosome. For example, FIG. 54A depicts a schematic of an engineered bacterium comprising, a circuit for butyrate production, a circuit for propionate production, and a circuit for production of one or more interleukins relevant to IBD. Fig. 54B depicts a schematic of an engineered bacterium comprising three circuits, a circuit for butyrate production, a circuit for GLP-2 expression and and a circuit for production of one or more interleukins relevant to IBD.

[093] FIG. 55 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[094] FIG. 56 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.

[095] FIG. 57 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 To1C (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.

[096] FIG. 58 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., 1pp, ompC, ompA, ompF, tolA, to1B, 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.

[097] FIG. 59 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).

[098] FIGs. 60A- 60C depict 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 (P
\ - araBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin.
The anti-toxin builds up in the recombinant bacterial cell, while TetR
prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed.
Since TetR
is not present to repress expression of the toxin, the toxin is expressed and kills the cell.
FIG. 60A 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. 60B 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. 60C 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.

[099] FIG. 61 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.

[0100] FIG. 62 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.

[0101] FIG. 63 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.

[0102] FIG. 64 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.

[0103] FIG. 65 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.

[0104] FIG. 66 depicts P-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 the tables (Pfnr1-5). 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 f3-galactosidase levels was analyzed by performing standard P-galactosidase colorimetric assays.

[0105] FIGs. 67A-67C depict a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (Pfnrs) and corresponding graphical data.
FIGs. 67A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (Pfnrs). LacZ encodes the P-galactosidase enzyme and is a common reporter gene in bacteria. FIG. 67B depicts FNR promoter activity as a function of P-galactosidase activity in 5YN340. 5YN340, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen.
Values for standard P-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. 67C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

[0106] FIGs. 68A-68D depict bar graphs, schematic, and dot blot, respectively, showing the structure or activity of reporter constructs. FIG. 68A and FIG.
68B depict bar graphs of reporter constructs activity. FIG. 68A depicts a graph of an ATC-inducible reporter construct expression and FIG. 68B 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 Ptet-GFP reporter construct or the nitric oxide inducible P
- nsr12.-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units. FIG. 68C depicts a schematic of the constructs.
FIG. 68D
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.

[0107] FIG. 69 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.

[0108] FIG. 70 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.

[0109] FIG. 71A and FIG. 71B depict a schematic diagrams of a wild-type clbA
construct (FIG. 71A) and a schematic diagram of a clbA knockout construct (FIG. 71B).

[0110] FIG. 72 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.

[0111] FIG. 73 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 (SC1): loop full ¨ glycerol stock, duration overnight, temperature 37 C, shaking at 250 rpm. Step 2 depicts the parameters for starter culture 2 (5C2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37 C, shaking at 250 rpm. Step 3 depicts the parameters for the production bioreactor: inoculum ¨ 5C2, 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 1X 10% glycerol/PBS, centrifugation, re-suspension 10%
glycerol/PBS.
Step 5 depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80 C.

[0112] Fig. 74 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.

[0113] Fig. 75A 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 +
10mM thymidine at 37C. The next day, cells were diluted 1:100 in 1 mL LB +
10mM
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 0D600 is measured every 5 minutes for minutes. This data shows that Nissle thyA auxotroph does not grow in environments lacking thymidine.

[0114] Fig. 75B 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.

[0115] Fig. 76 depicts a one non-limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti-toxin decays completely, and the cell dies.

[0116] Figs. 77A-77D depict schematics of non-limiting examples of the gene organization of plasmids, which function as a component of a bio safety system (Fig. 77A
and Fig. 77B), which also contains a chromosomal component (shown in Fig. 77C
and Fig. 77D). The bosafety plasmid system vector comprises Kid Toxin and R6K
minimal ori, dapA (Fig. 77A) and thyA (Fig. 77B) 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 (P0I2), and/or a third protein of interest (P0I3) are expressed from an inducible or constitutive promoter. Fig. 77C and Fig. 77D
depict schematics of the gene organization of the chromosomal component of a biosafety system. Fig. 77C 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.

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. 77A), then the chromosomal constructs shown in Fig. 77C and Fig. 77D are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in Fig. 77B), then the chromosomal constructs shown in Fig. 77C and Fig. 77D 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.

[0117] Fig. 78 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, pe1B-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.

[0118] Fig. 79 depicts the gene organization of exemplary construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.

[0119] Fig. 80A 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 anti-cancer molecules or immune modulatory effectors (POII) and a second set of one or more proteins of interest (P0I2), 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.

[0120] 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 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 Lad I 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.

[0121] Fig. 80B 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 Fnrs24Y
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. Bioinformatics tools for optimization of RBS are known in the art.

[0122] Fig. 80C depicts a strategy to fine-tune the expression of a Para-POI
construct by using a ribosome binding site optimization strategy.
Bioinformatics 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 Pthrs-POI constructs are maintained to allow for strong in vivo induction.

[0123] Fig. 81 depicts the gene organization of an exemplary construct, e.g., comprised in SYN-PKU401, comprising a cloned POI gene under the control of a Tet promoter sequence and a Tet repressor gene.

[0124] Fig. 82 depicts the gene organization of an exemplary construct comprising Lad I 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.

[0125] 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 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. P012 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. 77A, Fig.
77B, Fig.
77C, and Fig. 77D. In some embodiments, the construct is integrated into the genome at one or more locations described herein.

[0126] Fig. 83A, Fig. 83B, and Fig. 83C depict schematics of non-limiting examples of constructs for the expression of proteins of interest POI(s). Fig 83A 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 POI1 and/or a P012 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 P012 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. P012 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 P013 expression construct.

[0127] 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).

[0128] Fig. 84A 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.

[0129] Fig. 84B 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).

[0130] Fig. 85A depicts a schematic diagram of a wild-type clbA construct.

[0131] Fig. 85B depicts a schematic diagram of a clbA knockout construct.
Description of Embodiments

[0132] The present disclosure includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of reducing gut inflammation, enhancing gut barrier function, and/or treating or preventing autoimmune disorders. In some embodiments, the genetically engineered bacteria comprise at least one non-native gene and/or gene cassette for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the at least one gene and/or gene cassette is further operably linked to a regulatory region that is controlled by a transcription factor that is capable of sensing an inducing condition, e.g., a low-oxygen environment, the presence of ROS, or the presence of RNS. The genetically engineered bacteria are capable of producing the anti-inflammation and/or gut barrier function enhancer molecule(s) in inducing environments, e.g., in the gut. Thus, the genetically engineered bacteria and pharmaceutical compositions comprising those bacteria may be used to treat or prevent autoimmune disorders and/or diseases or conditions associated with gut inflammation and/or compromised gut barrier function, including IBD.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] A disease or condition associated with gut inflammation and/or compromised gut barrier function may be an autoimmune disorder. A disease or condition associated with gut inflammation and/or compromised gut barrier function may be co-morbid with an autoimmune disorder. As used herein, "autoimmune disorders"
include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylo sing 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 muco sal 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, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm &
testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis.

[0137] 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-(31, TGF-(32, 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-y, IL-113, 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., IPA), 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., IPA). 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 P13 gene.
Alternatively, an anti-inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., butyrate. These molecules may also be referred to as therapeutic molecules. In some instances, the "anti-inflammation molecules" and/or "gut barrier function enhancer molecules" are referred to herein as "effector molecules" or "therapeutic molecules" or "therapeutic polypeptides".

[0138] 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.

[0139] A "programmed or engineered microorganism" refers to a microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state to perform a specific function. Thus, a "programmed or engineered bacterial cell" or "programmed or engineered bacteria" refers to a bacterial cell or bacteria that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.

[0140] As used herein, the term "gene" refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. In one embodiment, a "gene" does not include regulatory sequences preceding and following the coding sequence. A "native gene" refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A "chimeric gene" refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature.

[0141] 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.

[0142] In some embodiments, the term "gene" or "gene sequence" is meant to refer to a nucleic acid sequence encoding any of the anti-inflammatory and gut barrier function enhancing molecules described herein, e.g., IL-2, IL-22, superoxide dismutase (SOD), kynurenine, GLP-2, GLP-1, IL-10, IL-27, TGF-01, TGF-02, N-acylphosphatidylethanolamines (NAPEs), elafin, and trefoil factor, as well as others. The nucleic acid sequence may comprise the entire gene sequence or a partial gene sequence encoding a functional molecule. The nucleic acid sequence may be a natural sequence or a synthetic sequence. The nucleic acid sequence may comprise a native or wild-type sequence or may comprise a modified sequence having one or more insertions, deletions, substitutions, or other modifications, for example, the nucleic acid sequence may be codon-optimized.

[0143] As used herein, a "heterologous" gene or "heterologous sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell and can be a native sequence (naturally found or expressed in the cell) or non-native sequence (not naturally found or expressed in the cell) and can be a natural or wild-type sequence or a variant, non-natural, or synthetic sequence.
"Heterologous gene" includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, the term "transgene" refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.

[0144] As used herein, a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a microorganism, 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 virus, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria or virus of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, "non-native" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature.
The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the genetically engineered microorganism of the disclosure comprises a gene that is operably linked to a promoter that is not associated with said gene in nature.
For example, in some embodiments, the genetically engineered bacteria disclosed herein comprise a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., an FNR responsive promoter (or other promoter disclosed herein) operably linked to an anti-inflammatory or gut barrier enhancer molecule. In some embodiments, the genetically engineered virus of the disclosure comprises a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., a promoter operably linked to a gene encoding an anti-inflammatory or gut barrier enhancer molecule.

[0145] As used herein, the term "coding region" refers to a nucleotide sequence that codes for a specific amino acid sequence. The term "regulatory sequence"
refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence.
Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, signal sequences, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR
responsive promoter or other promoter disclosed herein.

[0146] As used herein, a "gene cassette" or "operon" encoding a biosynthetic pathway refers to the two or more genes that are required to produce an anti-inflammatory or gut barrier enhancer molecule. 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.

[0147] A "butyrogenic gene cassette," "butyrate biosynthesis gene cassette,"
and "butyrate operon" are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. The genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria. A butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013). One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. A
butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiAl from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.
Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA
reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. The butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.

[0148] Likewise, a "propionate gene cassette" or "propionate operon" refers to a set of genes capable of producing propionate in a biosynthetic pathway.
Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. The genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria. In some embodiments, the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, e0, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA

dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyses the reduction of lactate to propionate.
Dehydration of (R)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA
by lactoyl-CoA dehydratase (LcdABC). Acrolyl-CoA is converted to propionyl-CoA
by acrolyl-CoA reductase (EtfA, AcrBC). In some embodiments, the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the actd gene from R. sphaeroides. This gene product catalyzes the NADPH-dependent acrylyl-CoA
reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides; Asao 2013). Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and actd.
In another embodiment, the homolog of AcuI in E coli, YhdH is used (see.e.g., Structure of Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004).
This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and 1pd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.

[0149] In another example of a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH).
Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering Escherichia coli for high-level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g. by the genetically engineered bacteria of the disclosure.
The SBM
pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm (methylmalonyl-CoA mutase) converts succinyl CoA to L-methylmalonylCoA, YgfD is a Sbm-interacting protein kinase with GTPase activity, ygfG
(methylmalonylCoA
decarboxylase) converts L-methylmalonylCoA into PropionylCoA, and ygfH
(propionyl-CoA/succinylCoA transferase) converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009). This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate.
Succinyl-CoA
is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134). There are three genes which encode methylmalonyl-CoA
carboxytransferase (mmdA, PFREUD 18870, bccp) which converts methylmalonyl-CoA

to propionyl-CoA.
[01501 The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate. One or more of the propionate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
[0151] An "acetate gene cassette" or "acetate operon" refers to a set of genes capable of producing acetate in a biosynthetic pathway. Bacteria "synthesize acetate from a number of carbon and energy sources," including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008). The genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria. Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994). Several bacteria, such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or CO2 + H2 into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art. The acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
[0152] Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.
[0153] Each gene or gene cassette may be operably linked to a promoter that is induced under low-oxygen conditions. "Operably linked" refers a nucleic acid sequence, e.g., a gene or gene cassette for producing an anti-inflammatory or gut barrier enhancer molecule, 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 "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding an anti-inflammatory or gut barrier enhancer molecule, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the anti-inflammatory or gut barrier enhancer molecule.
In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be "directly linked" to a regulatory sequence in a manner which allows expression of the gene. In another embodiment, a gene may be "indirectly linked" to a regulatory sequence in a manner which allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes. A regulatory region or sequence 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.
[0154] A "promoter" as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5' of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue-specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes:
inducible and constitutive. A "constitutive promoter" refers to a promoter that allows for continual transcription of the coding sequence or gene under its control.
[0155] "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, Ptac promoter, BBa J23100, a constitutive Escherichia coli GS 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 a32 promoter (e.g., htpG heat shock promoter (BBa J45504)), a constitutive Escherichia coli a70 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 K119000;
BBa K119001); 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 GA promoter (e.g., promoter veg (BBa K143013), promoter 43 (BBa K143013), PliaG (BBa K823000), PlepA (BBa K823002), Pveg (BBa K823003)), a constitutive Bacillus subtilis GB promoter (e.g., promoter ctc (BBa K143010), promoter gsiB (BBa K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa K112706), Pspv from Salmonella (BBa K112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa I712074; BBa I719005;
BBa J34814; BBa J64997; BBa K113010; BBa K113011; BBa K113012; BBa R0085;
BBa R0180; BBa R0181; BBa R0182; BBa R0183; BBa Z0251; BBa Z0252;
BBa Z0253)), and a bacteriophage 5P6 promoter (e.g., 5P6 promoter (BBa J64998)).
[0156] An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. An "inducible promoter" refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A "directly inducible promoter" refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An "indirectly inducible promoter" refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by "inducible promoter." Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. Examples of inducible promoters include, but are not limited to, an FNR
responsive promoter, a ParaC promoter, a ParaBAD promoter, and a PTetR
promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.
[0157] As used herein, "stably maintained" or "stable" bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a gene encoding one or more anti-inflammation and/or gut barrier enhancer molecule(s), which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated.
The stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium may be a genetically engineered bacterium comprising a gene encoding a encoding a payload, e.g., one or more anti-inflammation and/or gut barrier enhancer molecule(s), in which the plasmid or chromosome carrying the gene is stably maintained in the bacterium, such that the payload can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.
[0158] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.
[0159] As used herein, the term "plasmid" or "vector" refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell's genome. Plasmids are usually circular and capable of autonomous replication.
Plasmids may be low-copy, medium-copy, or high-copy, as is well known in the art.
Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding an anti-inflammatory or gut barrier enhancer molecule.

[0160] As used herein, the term "transform" or "transformation" refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically-stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.
[0161] The term "genetic modification," as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, base substitution, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising an anti-inflammatory or gut barrier enhancer molecule operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation.
[0162] As used herein, the term "genetic mutation" refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example, substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene.
The term "genetic mutation" is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene's polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.
[0163] As used herein, the term "transporter" is meant to refer to a mechanism, e.g., protein, proteins, or protein complex, for importing a molecule, e.g., amino acid, peptide (di-peptide, tri-peptide, polypeptide, etc), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.
[0164] As used herein, the phrase "exogenous environmental condition" or "exogenous environment signal" refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced. The phrase "exogenous environmental conditions" is meant to refer to 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 low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is specific to an inflammatory disease. In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels.
Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An "oxygen level-dependent promoter" or "oxygen level-dependent regulatory region" refers to a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.
[0165] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR (fumarate and nitrate reductase), ANR, and DNR.
Corresponding FNR-responsive promoters, ANR (anaerobic nitrate respiration)-responsive promoters, and DNR (dissimilatory nitrate respiration regulator)-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991;
Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1A.
[0166] 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 1A. Examples of transcription factors and responsive genes and regulatory regions Transcription Examples of responsive genes, Factor promoters, and/or regulatory regions:
FNR nirB, ydfZ, pdhR, focA, ndH, hlyE, narK, narX, narG, yfiD, tdcD
ANR arcDABC

DNR norb, norC
[0167] As used herein, a "tunable regulatory region" refers to a nucleic acid sequence under direct or indirect control of a transcription factor and which is capable of activating, repressing, derepressing, or otherwise controlling gene expression relative to levels of an inducer. In some embodiments, the tunable regulatory region comprises a promoter sequence. The inducer may be RNS, or other inducer described herein, and the tunable regulatory region may be a RNS-responsive regulatory region or other responsive regulatory region described herein. The tunable regulatory region may be operatively linked to a gene sequence(s) or gene cassette for the production of one or more payloads, e.g., a butyrogenic or other gene cassette or gene sequence(s). For example, in one specific embodiment, the tunable regulatory region is a RNS-derepressible regulatory region, and when RNS is present, a RNS-sensing transcription factor no longer binds to and/or represses the regulatory region, thereby permitting expression of the operatively linked gene or gene cassette. In this instance, the tunable regulatory region derepresses gene or gene cassette expression relative to RNS levels. Each gene or gene cassette may be operatively linked to a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one RNS.
[0168] In some embodiments, the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS). In some embodiments, exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut. In some embodiments, the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules.
[0169] In some embodiments, the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response). In some embodiments, the loss of exposure to an exogenous environmental condition (for example, in vivo) inhibits the activity of an inducible promoter, as the exogenous environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut). "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.
[0170] 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% 02, <160 ton 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 ton 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 1B 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% 02, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
Table 1B.
Compartment Oxygen Tension stomach -60 torr (e.g., 58 +/- 15 ton) duodenum and first part of -30 ton (e.g., 32 +/- 8 ton); -20% oxygen in jejunum ambient air Ileum (mid- small intestine) ¨10 torr; ¨6% oxygen in ambient air (e.g., 11 +/-ton) Distal sigmoid colon ¨ 3 ton (e.g., 3 +/- 1 torr) colon <2torr Lumen of cecum <1 ton tumor <32 ton (most tumors are <15 ton) [0171] "Microorganism" refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, e.g., Saccharomyces, and protozoa. In some aspects, the microorganism is engineered ("engineered microorganism") to produce one or more therpauetic molecules, e.g., an antinflammatory or barrier enhancer molecule. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus.
[0172] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria do not contain lipopolysaccharides (LPS). In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to certain strains belonging to the genus 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 lon gum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,Lactococcus lactis and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No.
6,203,797;
U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Non-pathogenic bacteria also include commensal bacteria, which are present in the indigenous microbiota of the gut. In one embodiment, the disclosure further includes non-pathogenic Saccharomyces, such as Saccharomyces boulardii. Naturally pathogenic bacteria may be genetically engineered to reduce or eliminate pathogenicity.
[0173] "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. In some embodiments, the probiotic bacteria are Gram-negative bacteria. In some embodiments, the probiotic bacteria are Gram-positive bacteria. 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, certain strains belonging to the genus Bifidobacteria, Escherichia Coli, Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, and Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797;
U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
[0174] 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).
[0175] As used herein, the term "auxotroph" or "auxotrophic" refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient) to support its growth. An "auxotrophic modification" is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient. As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival.
Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA).
[0176] As used herein, the terms "modulate" and "treat" a disease and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof. In another embodiment, "modulate" and "treat"
refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "modulate" and "treat" refer to inhibiting the progression of a 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, "modulate" and "treat" refer 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.
[0177] 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. Treating autoimmune disorders and/or diseases and conditions associated with gut inflammation and/or compromised gut barrier function may encompass reducing or eliminating excess inflammation and/or associated symptoms, and does not necessarily encompass the elimination of the underlying disease.
[0178] Treating the diseases described herein may encompass increasing levels of butyrate, increasing levels of acetate, increasing levels of butyrate and increasing GLP-2, IL-22, and/o rIL-10, and/or modulating levels of tryptophan and/or its metabolites (e.g., kynurenine), and/or providing any other anti-inflammation and/or gut barrier enhancer molecule and does not necessarily encompass the elimination of the underlying disease.
[0179] As used herein a "pharmaceutical composition" refers to a preparation of genetically engineered microorganism of the disclosure, e.g., genetically engineered bacteria or virus, with other components such as a physiologically suitable carrier and/or excipient.
[0180] 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 or viral compound. An adjuvant is included under these phrases.
[0181] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
Examples include, but are not limited to, calcium bicarbonate, sodium bicarbonate calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.
[0182] 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., inflammation, diarrhea.an autoimmune disorder. 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 an autoimmune a disorder and/or a disease or condition associated with gut inflammation and/or compromised gut barrier function. 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.

[0183] As used herein, the term "bacteriostatic" or "cytostatic" refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of recombinant bacterial cell of the disclosure.
[0184] As used herein, the term "bactericidal" refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.
[0185] As used herein, the term "toxin" refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure.
The term "toxin" is intended to include bacteriostatic proteins and bactericidal proteins.
The term "toxin" is intended to include, but not limited to, lytic proteins, bacteriocins (e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term "anti-toxin"
or "antitoxin," as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term anti-toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.
[0186] As used herein, "payload" refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus. In some embodiments, the payload is a therapeutic payload, e.g. and antiinflammatory or gut barrier enhancer molecule, e.g. butyrate, acetate, propionate, GLP-2, IL-10, IL-22, IL-2, other interleukins, and/or tryptophan and/or one or more of its metabolites.
In some embodiments, the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments the payload comprises an antibiotic resistance gene or genes. In some embodiments, the payload is encoded by a gene, multiple genes, gene cassette, or an operon. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads.
[0187] As used herein, the term "conventional treatment" or "conventional therapy" refers to treatment or therapy that is currently accepted, considered current standard of care, and/or used by most healthcare professionals for treating a disease or disorder associated with BCAA. It is different from alternative or complementary therapies, which are not as widely used.
[0188] 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.
[0189] 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.
[0190] 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. 0., 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, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His;
-Phe, Tyr, Trp, His; and -Asp, Glu.
[0191] 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 ic, k, a, y, 6, , and 11 constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either lc or k. Heavy chains are classified as y, 1,t, a, 6, or , 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.
[0192] As used herein, the term "antibody" or "antibodies"is meant to encompasses all variations of antibody and fragments thereof that possess one or more particular binding specificities. Thus, the term "antibody" or "antibodies" is meant to include full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single domain antibodies (sdAB, VHH framents), heavy chain antibodies (HCAb), nanobodies, diabodies, and minibodies. Antibodies can have more than one binding specificity, e.g., be bispecific. The term "antibody" is also meant to include so-called antibody mimetics. Antibody mimetics refers to small molecules, e.g., 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure. Antibody mimetics, include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B
crystalline), Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from Sulfolobus acidocaldarius), Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif), Fynomers (SH3 domain of Fyn), Kunitz domain peptides Kunitz domains of various protease inhibitors), Ecallantide (Kalbitor), and Monobodies. In certain aspects, the term "antibody" or "antibodies" is meant to refer to a single chain antibody(ies), single domain antibody(ies), and camelid antibody(ies). Utility of antibodies in the treatment of cancer and additional anti cancer antibodies can for example be found in Scott et al., Antibody Therapy for Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference in its entirety.
[0193] 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.
[0194] In some embodiments, the antibodies expressed by the engineered microorganisms are bispecfic. In certain embodiments, a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope.
Antigen-binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Monomeric single-chain diabodies (scDb) are readily assembled in bacterial and mammalian cells and show improved stability under physiological conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001) 14 (10): 815-823 (describes optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies).
[0195] 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.

[0196] 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.
[0197] As used herein the term "codon-optimized" refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A "codon-optimized sequence" refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA
molecule transcribed from the coding sequence, or to improve transcription of a coding sequence.
Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism.
Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
[0198] As used herein, the terms "secretion system" or "secretion protein"
refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g., polypeptide from the microbial, e.g., bacterial cytoplasm.
The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g.,HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems.
Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT
secretion systems. In some embodiments, the polypeptide to be secreted include a "secretion tag" of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antinflammatory or barrier enhancer molecule(s) into the extracellular milieu.
In some embodiments, the secretion system involves the generation of a "leaky" or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, 1pp, ompC, ompA, ompF, tolA, to1B, pal, degS, degP, and nlpl.
Lpp functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. To1A-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g., selected from 1pp, ompA, ompA, ompF, tolA, to1B, and pal genes.
In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from 1pp, ompA, ompA, ompF, tolA, to1B, pal, degS, degP, and nlpl genes.
[0199] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0200] 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.
[0201] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Bacteria [0202] The genetically engineered microorganisms, or programmed microorganisms, such as genetically engineered bacteria of the disclosure are capable of producing one or more non-native anti-inflammation and/or gut barrier function enhancer molecules. 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. 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 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, 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 cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT, 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. 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, Clostridium clusters IV and XIVa of Firmicutes (including species of Eubacterium), Roseburia, Faecalibacterium, Enterobacter, Faecalibacterium prausnitzii, Clostridium difficile, Subdoligranulum, Clostridium sporogenes, Campylobacter jejuni, Clostridium saccharolyticum, Klebsiella, Citrobacter, Pseudobutyrivibrio, and Ruminococcus. In certain embodiments, the the genetically engineered bacteria are selected from Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis [0203] In some embodiments, the genetically engineered bacterium is a Gram-positive bacterium, e.g., Clostridium, that is naturally capable of producing high levels of butyrate. In some embodiments, the genetically engineered bacterium is selected from the group consisting of C. butyricum ZJUCB, C. butyricum S21, C. thermobutyricum ATCC
49875, C. beijerinckii, C. populeti ATCC 35295, C. tyrobutyricum JM1, C.
tyrobutyricum CIP 1-776, C. tyrobutyricum ATCC 25755, C. tyrobutyricum CNRZ 596, and C.
tyrobutyricum ZJU 8235. In some embodiments, the genetically engineered bacterium is C. butyricum CBM588, a probiotic bacterium that is highly amenable to protein secretion and has demonstrated efficacy in treating IBD (Kanai et al., 2015). In some embodiments, the genetically engineered bacterium is Bacillus, a probiotic bacterium that is highly genetically tractable and has been a popular chassis for industrial protein production; in some embodiments, the bacterium has highly active secretion and/or no toxic byproducts (Cutting, 2011).
[0204] In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell.
In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell.
In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell.
In one embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell.
[0205] 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). In some embodiments, the genetically engineered bacteria are E. coli Nissle and are naturally capable of promoting tight junctions and gut barrier function. In some embodiments, the genetically engineered bacteria are E. coli and are highly amenable to recombinant protein technologies.
[0206] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. It is known, for example, that the clostridial butyrogenic pathway genes are widespread in the genome-sequenced clostridia and related species (Aboulnaga et al., 2013). Furthermore, genes from one or more different species of bacteria can be introduced into one another, e.g., the butyrogenic genes fromPeptoclostridium difficile have been expressed in Escherichia coli (Aboulnaga et al., 2013).
[0207] . In one embodiment, the recombinant bacterial cell does not colonize the subject having the disorder. Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria. In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention, e.g. as described herein.
[0208] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells disclosed herein.
[0209] In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein.
[0210] In some embodiments, the genetically engineered bacteria comprising an anti-inflammatory or gut barrier enhancer molecule further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
[0211] In some embodiments, the genetically engineered bacteria is an auxotroph comprising gene sequence encoding an anti-inflammatory or gut barrier enhancer molecule and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.
[0212] In some embodiments of the above described genetically engineered bacteria, the gene encoding an anti-inflammatory or gut barrier enhancer molecule is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an anti-inflammatory or gut barrier enhancer molecule is present in the bacterial chromosome. In some embodiments, a gene sequence encoding a secretion protein or protein complex, such as any of the secretion systems disclosed herein, for secreting a biomolecule (e.g. an anti-inflammatory or gut barrier enhancer molecule), is present on a plasmid in the bacterium. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome.
Anti-inflammation and/or gut barrier function enhancer molecules [0213] The genetically engineered bacteria comprise one or more gene sequence(s) and/or gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene sequence(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene sequences are multiple copies of the same gene. In some emodiments, the two or more gene sequences are sequences encoding different genes. In some emodiments, the two or more gene sequences are sequences encoding multiple copies of one or more different genes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene cassettes are multiple copies of the same gene cassette. In some emodiments, the two or more gene cassettes are different gene cassettes for producing either the same or different anti-inflammation and/or gut barrier function enhancer molecule(s). In some emodiments, the two or more gene cassettes are gene cassettes for producing multiple copies of one or more different anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the anti-inflammation and/or gut barrier function enhancer molecule is selected from the group consisting of a short-chain fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP-1, IL-10 (human or viral), IL-27, TGF-(31, TGF-(32, N-acylphosphatidylethanolamines (NAPEs), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, kynurenine, typtophan metabolite, indole, indole metabolite, a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN-y, IL-113, IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2, AHR agonist (e.g., indole acetic acid, indole-3-aldehyde, and indole), PXR agonist (e.g., IPA), HDAC inhibitor (e.g., butyrate), GPR41 and/or GPR43 activator (e.g., butyrate and/or propionate and/or acetate), activator (e.g., butyrate), inhibitor of NF-kappaB signaling (e.g., butyrate), modulator of PPARgamma (e.g., butyrate), activator of AMPK signaling (e.g., acetate), modulator of GLP-1 secretion, and hydroxyl radical scavengers and antioxidants (e.g., IPA).
A
molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2. Alternatively, a molecule may be both anti-inflammatory and gut barrier function enhancing.

[0214] In some embodiments, the genetically engineered bacteria of the invention express one or more anti-inflammation and/or gut barrier function enhancer molecule(s) that is encoded by a single gene, e.g., the molecule is elafin and encoded by the PI3 gene, or the molecule is interleukin-10 and encoded by the IL10 gene. In alternate embodiments, the genetically engineered bacteria of the invention encode one or more an anti-inflammation and/or gut barrier function enhancer molecule(s), e.g., butyrate, that is synthesized by a biosynthetic pathway requiring multiple genes.
[0215] The one or more gene sequence(s) and/or gene cassette(s) may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing expression of the anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the gene sequence(s)or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the butyrate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed from a plasmid in the genetically engineered bacteria.
In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle:
malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 52 for exemplary insertion sites). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
Short chain Fatty Acids and Tryptophan Metabolites [0216] One strategy in the treatment, prevention, and/or management of inflammatory bowel disorders may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti-inflammatory effectors.
[0217] For example, leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate, and those derived from tryptophan metabolism. These metabolites have been shown to play a major role in the prevention of inflammatory disease. As such one approach in the treatment, prevention, and/or management of gut barrier health may be to provide a treatment which contains one or more of such metabolites.
[0218] For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity (e.g., as reviewed in Thorburn et al., Diet, Metabolites, and "Western-Lifestyle" Inflammatory Diseases; Immunity Volume 40, Issue 6, 19 June 2014, Pages 833-842). (A) SCFA-induced promotion of mucus by gut epithelial cells, possibly through signaling through metabolite sensing GPCRs;
(B) SCFA-induced secretion of IgA by B cells; (C) SCFA-induced promotion of tissue repair and wound healing; (D) SCFA-induced promotion of Treg cell development in the gut in a process that presumably facilitates immunological tolerance; (E) SCFA-mediated enhancement of epithelial integrity in a process dependent on inflammasome activation (e.g., via NALP3) and IL-18 production; and (F) anti-inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, 11-6, and IFN-gamma), and inhibition of NF-KB. Many of these actions of SCFAs in gut homeostatis can be ascribed to GPR43 and GPR109A, which are expressed by the colonic epithelium, by inflammatory leukocytes (e.g. neutrophils and marcophages) and by Treg cells. These receptors signal through G
proteins, coupled to MAPK, PI3K and mTOR, as well as a separate arrestin-pathway, leading to NFkappa B inhibition. Other effects can be ascribed to SCFA-mediated HDAC
inhibition, e.g. butyrate, which may regulate macrophage function and promote TReg cells.
[0219] In addition, a number of tryptophan metabolites, including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehyde, 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 Th17 cell activity, and the maintenance of intraepithelial lymphocytes and RORyt+ innate lymphoid cells.
[0220] 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.
[0221] In addition, some indole metabolites, e.g., indole 3-propionic acid (IPA), 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 expression to promote homeostasis and gut barrier health.
[0222] Thus, in some embodiments, the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryprophan metabolites.
Acetate [0223] In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and are capable of producing acetate. The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. In other embodiments, the bacteria eomprise an endogenous acetate biosynthetic gene or gene cassette and naturally produce acetate. Unmodified bacteria comprising acetate biosynthesis genes are known in the art and are capable of consuming various substrates to produce acetate under aerobic and/or anaerobic conditions (see, e.g., Ragsdale, 2008), and these endogenous acetate biosynthesis pathways may be a source of genes for the genetically engineered bacteria of the invention. In some embodiments, the genetically engineered bacteria of the invention comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced. In some embodiments, the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli. In some embodiments, the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and/or The rmoacetogenium. The genetically engineered bacteria may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, the genetically engineered bacteria comprise a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing acetate. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid.
[0224] In E. coli Nissle, acetate is generated as an end product of fermentation. In E coli, glucose fermentation occurs in two steps, (1) the glycolysis reactions and (2) the NADH recycling reactions, i.e. these reactions re-oxidize the NAD+ generated during the fermentation process. E. coli employs the "mixed acid" fermentation pathway (see, e.g., FIG 25). Through the "mixed acid" pathway, E coli generates several alternative end products and in variable amounts (e.g., lactate, acetate, formate, succinate, ethanol, carbon dioxide, and hydrogen) though various arms of the fermentation pathway, e.g., as shown in FIG. 25. Without wishing to be bound by theory, prevention or reduction of flux through one or more metabolic arm(s) generating metabolites other than acetate, e.g.
through mutation, deletion and/or inhibition of one or more gene(s) encoding key enzymes in these metabolic arms, results in an increase in production of acetate for NAD recycling.
As disclosed herein, e.g., in Example 20, deletions in gene(s) encoding such enzymes increase acetate production. Such enzymes include fumarate reductase (encoded by the frd genes), lactate dehydrogenase (encoded by the ldh gene), and aldehyde-alcohol dehydrogenase (encoded by the adhE gene).
[0225] LdhA is a soluble NAD-linked lactate dehydrogenase (LDH) that is specific for the production of D-lactate and is a homotetramer and shows positive homotropic cooperativity under higher pH conditions. E. coli carrying ldhA
mutations show no observable growth defect and can still ferment sugars to a variety of products other than lactate.
[0226] In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous ldhA gene.
[0227] AdhE is a homopolymeric protein with three catalytic functions: alcohol dehydrogenase, coenzyme A-dependent acetaldehyde dehydrogenase, and pyruvate formate-lyase deactivase. During fermentation, AdhE has catalyzes two steps towards the generation of ethanol: (1) the reduction of acetyl-CoA to acetaldehyde and (2) the reduction of acetaldehyde to to ethanol. Deletion of adhE has been employed to enhance production of certain metabolites inducstrially, including succinate, D-lactate, and polyhydroxyalkanoates (Singh et al, Manipulating redox and ATP balancing for improved production of succinate in E. coli.; Metab Eng. 2011 Jan;13(1):76-81; Zhou et al., Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli, Curr Microbiol. 2011 Mar;62(3):981-9; Jian et al., Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways, Appl Microbiol Biotechnol. 2010 Aug;87(6):2247-56).
[0228] In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous adhE gene.
[0229] The fumarate reductase enzyme complex, encoded by the frdABCD
operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. FrdA is one of two catalytic subunits in the four subunit fumarate reductase complex. FrdB is the second catalytic subinut of the complex.
FrdC and FrdD are two integral membrane protein components of the fumarate reductase complex. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA gene.

[0230] In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE
genes.
[0231] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0232] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding one or more enzyme(s) which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate.
[0233] Phosphate acetyltransferase (Pta) catalyzes the reversible conversion between acetyl-CoA and acetylphosphate, a step in the metabolism of acetate (Campos-Bermudez et al., Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation; FEBS J.

Apr;277(8):1957-66). Both pyruvate and phosphoenolpyruvate activate the enzyme in the direction of acetylphosphate synthesis and inhibit the enzyme in the direction of acetyl-CoA synthesis. The acetate formation from acetyl-CoA I pathway has been the target of metabolic engineering to reduce the flux to acetate and increase the production of commercially desired end products (see, e.g., Singh, et al., Manipulating redox and ATP
balancing for improved production of succinate in E. coli; Metab Eng. 2011 Jan;13(1):76-81). A pta mutant does not grow on acetate as the sole source of carbon (Brown et al., The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli;
J Gen Microbiol. 1977 Oct;102(2):327-36).
[0234] In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the gentically engineered bacteria produce butyrate.In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene and also in one or more endogenous genes selected from the ldhA gene, the frdA gene and the adhE
gene. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutationand/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the gentically engineered bacteris produce butyrate.
[0235] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
Butyrate [0236] In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 2 and Table 3).
Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from Peptoclostridium difficile, e.g., Peptoclostridium difficile strain 630: bcd2, e1J133, etfA3, thiAl, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate.
Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk. In some embodiments, the genetically engineered bacteria comprise a combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate. For example, in some embodiments, the genetically engineered bacteria comprise bcd2, etf733, effA3, and thiAl from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus, a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli).

Thus a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.n some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
[0237] In some embodiments, additional genes may be mutated or knocked out, to further increase the levels of butyrate production. Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0238] Table 2 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.
Table 2. Exemplary Butyrate Cassette Sequences Description Sequence ATGGATTTAAATTCTAAAAAATATCAGATGCTTAAAGAGCTATATGTAAG
CTTCGCTGAAAATGAAGTTAAACCTTTAGCAACAGAACTTGATGAAGAAG
AAAGATTTCCTTATGAAACAGTGGAAAAAATGGCAAAAGCAGGAATGATG
GGTATACCATATCCAAAAGAATATGGTGGAGAAGGTGGAGACACTGTAGG
ATATATAATGGCAGTTGAAGAATTGTCTAGAGTTTGTGGTACTACAGGAG
TTATATTATCAGCTCATACATCTCTTGGCTCATGGCCTATATATCAATAT
GGTAATGAAGAACAAAAACAAAAATTCTTAAGACCACTAGCAAGTGGAGA
AAAATTAGGAGCATTTGGTCTTACTGAGCCTAATGCTGGTACAGATGCGT
bcd2 CTGGCCAACAAACAACTGCTGTTTTAGACGGGGATGAATACATACTTAAT
SEQ ID NO: 1 GGCTCAAAAATATTTATAACAAACGCAATAGCTGGTGACATATATGTAGT
AATGGCAATGACTGATAAATCTAAGGGGAACAAAGGAATATCAGCATTTA
TAGTTGAAAAAGGAACTCCTGGGTTTAGCTTTGGAGTTAAAGAAAAGAAA
ATGGGTATAAGAGGTTCAGCTACGAGTGAATTAATATTTGAGGATTGCAG
AATACCTAAAGAAAATTTACTTGGAAAAGAAGGTCAAGGATTTAAGATAG
CAATGTCTACTCTTGATGGTGGTAGAATTGGTATAGCTGCACAAGCTTTA
GGTTTAGCACAAGGTGCTCTTGATGAAACTGTTAAATATGTAAAAGAAAG
AGTACAATTTGGTAGACCATTATCAAAATTCCAAAATACACAATTCCAAT
TAGCTGATATGGAAGTTAAGGTACAAGCGGCTAGACACCTTGTATATCAA

Description Sequence GCAGCTATAAATAAAGACT TAGGAAAACCT TAT GGAGTAGAAGCAGCAAT
GGCAAAAT TAT T T GCAGC T GAAACAGC TAT GGAAGT TACTACAAAAGCTG
TACAACT T CAT GGAGGATAT GGATACAC T CGT GAC TAT CCAGTAGAAAGA
AT GAT GAGAGAT GC TAAGATAAC T GAAATATAT GAAGGAAC TAGT GAAGT
TCAAAGAATGGT TAT T T CAGGAAAAC TAT TAAAATAG
AT GAATATAGT CGT T TGTATAAAACAAGT TCCAGATACAACAGAAGT TAA
AC TAGAT CC TAATACAGGTAC T T TAAT TAGAGATGGAGTACCAAGTATAA
TAAACCC T GAT GATAAAGCAGGT T TAGAAGAAGCTATAAAAT TAAAAGAA
GAAAT GGGT GC T CAT GTAAC T GT TATAACAAT GGGACC T CC T CAAGCAGA
TAT GGC T T TAAAAGAAGCT T TAGCAATGGGTGCAGATAGAGGTATAT TAT
TAACAGATAGAGCAT T T GCGGGT GC T GATAC T TGGGCAACT T CAT CAGCA
T TAGCAGGAGCAT TAAAAAATATAGAT T T TGATAT TATAATAGCTGGAAG
etfB3 ACAGGCGATAGATGGAGATACTGCACAAGT TGGACCTCAAATAGCTGAAC
SEQ ID NO: 2 AT T TAAATCT T CCAT CAATAACATAT GC T GAAGAAATAAAAAC T GAAGGT
GAATATGTAT TAGTAAAAAGACAAT T TGAAGAT T GT TGCCATGACT TAAA
AGT TAAAATGCCATGCCT TATAACAACTCT TAAAGATATGAACACACCAA
GATACATGAAAGT T GGAAGAATATAT GAT GC T T T CGAAAAT GAT GTAGTA
GAAACATGGACTGTAAAAGATATAGAAGT TGACCCT TCTAAT T TAGGTCT
TAAAGGT TCTCCAACTAGTGTAT T TAAAT CAT T TACAAAATCAGT TAAAC
CAGCTGGTACAATATACAATGAAGATGCGAAAACATCAGCTGGAAT TAT C
ATAGATAAAT TAAAAGAGAAGTATATCATATAA
AT GGGTAACGT T T TAGTAGTAATAGAACAAAGAGAAAATGTAAT TCAAAC
T GT TTCTT TAGAAT TACTAGGAAAGGCTACAGAAATAGCAAAAGAT TAT G
ATACAAAAGT TTCTGCAT TACT T T TAGGTAGTAAGGTAGAAGGT T TAATA
GATACAT TAGCACAC TAT GGT GCAGAT GAGGTAATAGTAGTAGAT GAT GA
AGCT T TAGCAGTGTATACAACTGAACCATATACAAAAGCAGCT TAT GAAG
CAATAAAAGCAGCTGACCCTATAGT TGTAT TAT T TGGTGCAACT TCAATA
GGTAGAGAT T TAGCGCCTAGAGT TTC T GC TAGAATACATACAGGT C T TAC
T GC T GAC T GTACAGGT C T TGCAGTAGCTGAAGATACAAAAT TAT TAT TAA
TGACAAGACCTGCCT T TGGTGGAAATATAATGGCAACAATAGT T TGTAAA
GAT T T CAGACC T CAAAT GT C TACAGT TAGACCAGGGGT TAT GAAGAAAAA
etfA3 T GAACC T GAT GAAAC TAAAGAAGC T GTAAT TAACCGT T TCAAGGTAGAAT
SEQ ID NO: 3 T TAAT GAT GC T GATAAAT TAGT TCAAGT TGTACAAGTAATAAAAGAAGCT
AAAAAACAAGT TAAAATAGAAGAT GC TAAGATAT TAGT TTCT GC T GGACG
TGGAATGGGTGGAAAAGAAAACT TAGACATACT T TAT GAAT TAGCTGAAA
T TATAGGTGGAGAAGT TTCTGGT TCTCGTGCCACTATAGATGCAGGT TGG
T TAGATAAAGCAAGACAAGT TGGTCAAACTGGTAAAACTGTAAGACCAGA
CC T T TATATAGCAT GT GGTATAT C T GGAGCAATACAACATATAGC T GGTA
T GGAAGAT GC T GAGT T TATAGT T GC TATAAATAAAAAT CCAGAAGC T CCA
ATAT T TAAATAT GC T GAT GT TGGTATAGT T GGAGAT GT T CATAAAGT GC T
TCCAGAACT TAT CAGT CAGT TAAGT GT TGCAAAAGAAAAAGGTGAAGT T T
TAGCTAACTAA
AT GAGAGAAGTAGTAAT TGCCAGTGCAGCTAGAACAGCAGTAGGAAGT T T
TGGAGGAGCAT T TAAATCAGT T TCAGCGGTAGAGT TAGGGGTAACAGCAG
thiAl CTAAAGAAGCTATAAAAAGAGCTAACATAACTCCAGATATGATAGATGAA
SEQ ID NO: 4 TCTCTTT TAGGGGGAGTACT TACAGCAGGTCT TGGACAAAATATAGCAAG
ACAAATAGCAT TAGGAGCAGGAATACCAGTAGAAAAACCAGC TAT GAC TA

Description Sequence TAAATATAGTTTGTGGTTCTGGATTAAGATCTGTTTCAATGGCATCTCAA
CTTATAGCATTAGGTGATGCTGATATAATGTTAGTTGGTGGAGCTGAAAA
CATGAGTATGTCTCCTTATTTAGTACCAAGTGCGAGATATGGTGCAAGAA
TGGGTGATGCTGCTTTTGTTGATTCAATGATAAAAGATGGATTATCAGAC
ATATTTAATAACTATCACATGGGTATTACTGCTGAAAACATAGCAGAGCA
ATGGAATATAACTAGAGAAGAACAAGATGAATTAGCTCTTGCAAGTCAAA
ATAAAGCTGAAAAAGCTCAAGCTGAAGGAAAATTTGATGAAGAAATAGTT
CCTGTTGTTATAAAAGGAAGAAAAGGTGACACTGTAGTAGATAAAGATGA
ATATATTAAGCCTGGCACTACAATGGAGAAACTTGCTAAGTTAAGACCTG
CATTTAAAAAAGATGGAACAGTTACTGCTGGTAATGCATCAGGAATAAAT
GATGGTGCTGCTATGTTAGTAGTAATGGCTAAAGAAAAAGCTGAAGAACT
AGGAATAGAGCCTCTTGCAACTATAGTTTCTTATGGAACAGCTGGTGTTG
ACCCTAAAATAATGGGATATGGACCAGTTCCAGCAACTAAAAAAGCTTTA
GAAGCTGCTAATATGACTATTGAAGATATAGATTTAGTTGAAGCTAATGA
GGCATTTGCTGCCCAATCTGTAGCTGTAATAAGAGACTTAAATATAGATA
TGAATAAAGTTAATGTTAATGGTGGAGCAATAGCTATAGGACATCCAATA
GGATGCTCAGGAGCAAGAATACTTACTACACTTTTATATGAAATGAAGAG
AAGAGATGCTAAAACTGGTCTTGCTACACTTTGTATAGGCGGTGGAATGG
GAACTACTTTAATAGTTAAGAGATAG
ATGAAATTAGCTGTAATAGGTAGTGGAACTATGGGAAGTGGTATTGTACA
AACTTTTGCAAGTTGTGGACATGATGTATGTTTAAAGAGTAGAACTCAAG
GTGCTATAGATAAATGTTTAGCTTTATTAGATAAAAATTTAACTAAGTTA
GTTACTAAGGGAAAAATGGATGAAGCTACAAAAGCAGAAATATTAAGTCA
TGTTAGTTCAACTACTAATTATGAAGATTTAAAAGATATGGATTTAATAA
TAGAAGCATCTGTAGAAGACATGAATATAAAGAAAGATGTTTTCAAGTTA
CTAGATGAATTATGTAAAGAAGATACTATCTTGGCAACAAATACTTCATC
hbd ATTATCTATAACAGAAATAGCTTCTTCTACTAAGCGCCCAGATAAAGTTA
TAGGAATGCATTTCTTTAATCCAGTTCCTATGATGAAATTAGTTGAAGTT
SEQ ID NO: 5 ATAAGTGGTCAGTTAACATCAAAAGTTACTTTTGATACAGTATTTGAATT
ATCTAAGAGTATCAATAAAGTACCAGTAGATGTATCTGAATCTCCTGGAT
TTGTAGTAAATAGAATACTTATACCTATGATAAATGAAGCTGTTGGTATA
TATGCAGATGGTGTTGCAAGTAAAGAAGAAATAGATGAAGCTATGAAATT
AGGAGCAAACCATCCAATGGGACCACTAGCATTAGGTGATTTAATCGGAT
TAGATGTTGTTTTAGCTATAATGAACGTTTTATATACTGAATTTGGAGAT
ACTAAATATAGACCTCATCCACTTTTAGCTAAAATGGTTAGAGCTAATCA
ATTAGGAAGAAAAACTAAGATAGGATTCTATGATTATAATAAATAA
ATGAGTACAAGTGATGTTAAAGTTTATGAGAATGTAGCTGTTGAAGTAGA
TGGAAATATATGTACAGTGAAAATGAATAGACCTAAAGCCCTTAATGCAA
TAAATTCAAAGACTTTAGAAGAACTTTATGAAGTATTTGTAGATATTAAT
AATGATGAAACTATTGATGTTGTAATATTGACAGGGGAAGGAAAGGCATT
TGTAGCTGGAGCAGATATTGCATACATGAAAGATTTAGATGCTGTAGCTG
crt2 CTAAAGATTTTAGTATCTTAGGAGCAAAAGCTTTTGGAGAAATAGAAAAT
SEQ ID NO: 6 AGTAAAAAAGTAGTGATAGCTGCTGTAAACGGATTTGCTTTAGGTGGAGG
ATGTGAACTTGCAATGGCATGTGATATAAGAATTGCATCTGCTAAAGCTA
AATTTGGTCAGCCAGAAGTAACTCTTGGAATAACTCCAGGATATGGAGGA
ACTCAAAGGCTTACAAGATTGGTTGGAATGGCAAAAGCAAAAGAATTAAT
CTTTACAGGTCAAGTTATAAAAGCTGATGAAGCTGAAAAAATAGGGCTAG

Description Sequence TAAATAGAGTCGT TGAGCCAGACAT TI TAATAGAAGAAGT TGAGAAAT TA
GC TAAGATAATAGC TAAAAAT GC T CAGC T TGCAGT TAGATACTCTAAAGA
AGCAATACAACT T GGT GC T CAAAC T GATATAAATAC T GGAATAGATATAG
AATCTAAT T TAT T T GGT CT T T GT TTTTCAACTAAAGACCAAAAAGAAGGA
AT GT CAGC T T TCGT TGAAAAGAGAGAAGCTAACT T TATAAAAGGGTAA
AT GAGAAGT II TGAAGAAGTAAT TAAGT T TGCAAAAGAAAGAGGACCTAA
AAC TATAT CAGTAGCAT GT TGCCAAGATAAAGAAGT II TAATGGCAGT TG
AAATGGCTAGAAAAGAAAAAATAGCAAATGCCAT II TAGTAGGAGATATA
GAAAAGACTAAAGAAAT TGCAAAAAGCATAGACATGGATATCGAAAAT TA
TGAACTGATAGATATAAAAGAT T TAGCAGAAGCAT C T C TAAAAT C T GT TG
AAT TAGT T TCACAAGGAAAAGCCGACATGGTAATGAAAGGCT TAGTAGAC
ACATCAATAATACTAAAAGCAGT II TAAATAAAGAAGTAGGTCT TAGAAC
TGGAAATGTAT TAAGTCACGTAGCAGTAT T T GAT GTAGAGGGATAT GATA
GAT TAT T T T T CGTAAC T GACGCAGC TAT GAAC T TAGC T CC T GATACAAAT
pbt AC TAAAAAGCAAAT CATAGAAAAT GC T TGCACAGTAGCACAT T CAT TAGA
SEQ ID NO: 7 TATAAGTGAACCAAAAGT T GC T GCAATAT GCGCAAAAGAAAAAGTAAAT C
CAAAAATGAAAGATACAGT TGAAGCTAAAGAACTAGAAGAAATGTATGAA
AGAGGAGAAATCAAAGGT TGTATGGT TGGTGGGCCTTTTGCAAT TGATAA
TGCAGTATCT T TAGAAGCAGC TAAACATAAAGGTATAAAT CAT CC T GTAG
CAGGACGAGCTGATATAT TAT TAGCCCCAGATAT TGAAGGTGGTAACATA
T TATATAAAGCT T TGGTAT TCTTCTCAAAATCAAAAAATGCAGGAGT TAT
AGT TGGGGCTAAAGCACCAATAATAT TAACT TCTAGAGCAGACAGTGAAG
AAACTAAACTAAACTCAATAGCT T TAGGT GT T T TAATGGCAGCAAAGGCA
TAA
AT GAGCAAAATAT T TAAAATCT TAACAATAAAT CC T GGT TCGACATCAAC
TAAAATAGCTGTAT T TGATAATGAGGAT T TAGTAT T TGAAAAAACT T TAA
GACAT TCT T CAGAAGAAATAGGAAAATAT GAGAAGGT GT C T GACCAAT T T
GAAT T TCGTAAACAAGTAATAGAAGAAGCTCTAAAAGAAGGTGGAGTAAA
AACATCTGAAT TAGAT GC T GTAGTAGGTAGAGGAGGAC T TCT TAAACC TA
TAAAAGGTGGTACT TAT T CAGTAAGT GC T GC TAT GAT TGAAGAT T TAAAA
GT GGGAGT TI TAGGAGAACACGCT TCAAACCTAGGTGGAATAATAGCAAA
ACAAATAGGT GAAGAAGTAAAT GT T CC T T CATACATAGTAGACCC T GT TG
T TGTAGATGAAT TAGAAGAT GI T GC TAGAAT TTCTGGTATGCCTGAAATA
AGTAGAGCAAGT GTAGTACAT GC T T TAAATCAAAAGGCAATAGCAAGAAG
buk ATAT GC TAGAGAAATAAACAAGAAATAT GAAGATATAAAT C T TATAGT TG
SEQ ID NO: 8 CACACATGGGTGGAGGAGT T TC T GT TGGAGCTCATAAAAATGGTAAAATA
GTAGAT GT TGCAAACGCAT TAGATGGAGAAGGACCTTTCTCTCCAGAAAG
AAGTGGTGGACTACCAGTAGGTGCAT TAGTAAAAAT GT GC T T TAGTGGAA
AATATACTCAAGATGAAAT TAAAAAGAAAATAAAAGGTAAT GGCGGAC TA
GI TGCATACT TAAACAC TAAT GAT GC TAGAGAAGT TGAAGAAAGAAT T GA
AGC T GGT GAT GAAAAAGC TAAAT TAGTATAT GAAGC TAT GGCATAT CAAA
TCTC TAAAGAAATAGGAGC TAGT GC T GCAGT TCT TAAGGGAGATGTAAAA
GCAATAT TAT TAACTGGTGGAATCGCATAT T CAAAAAT GT T TACAGAAAT
GAT TGCAGATAGAGT TAAAT T TATAGCAGATGTAAAAGT T TAT CCAGGT G
AAGAT GAAAT GAT TGCAT TAGCTCAAGGTGGACT TAGAGT T T TAACTGGT
GAAGAAGAGGCTCAAGT T TAT GATAAC TAA

Description Sequence ATGATCGTAAAACCTATGGTACGCAACAATATCTGCCTGAACGCCCATCC
TCAGGGCTGCAAGAAGGGAGTGGAAGATCAGATTGAATATACCAAGAAAC
GCATTACCGCAGAAGTCAAAGCTGGCGCAAAAGCTCCAAAAAACGTTCTG
GTGCTTGGCTGCTCAAATGGTTACGGCCTGGCGAGCCGCATTACTGCTGC
GTTCGGATACGGGGCTGCGACCATCGGCGTGTCCTTTGAAAAAGCGGGTT
CAGAAACCAAATATGGTACACCGGGATGGTACAATAATTTGGCATTTGAT
GAAGCGGCAAAACGCGAGGGTCTTTATAGCGTGACGATCGACGGCGATGC
GTTTTCAGACGAGATCAAGGCCCAGGTAATTGAGGAAGCCAAAAAAAAAG
GTATCAAATTTGATCTGATCGTATACAGCTTGGCCAGCCCAGTACGTACT
GATCCTGATACAGGTATCATGCACAAAAGCGTTTTGAAACCCTTTGGAAA
AACGTTCACAGGCAAAACAGTAGATCCGTTTACTGGCGAGCTGAAGGAAA
ter TCTCCGCGGAACCAGCAAATGACGAGGAAGCAGCCGCCACTGTTAAAGTT
SEQ ID NO: 9 ATGGGGGGTGAAGATTGGGAACGTTGGATTAAGCAGCTGTCGAAGGAAGG
CCTCTTAGAAGAAGGCTGTATTACCTTGGCCTATAGTTATATTGGCCCTG
AAGCTACCCAAGCTTTGTACCGTAAAGGCACAATCGGCAAGGCCAAAGAA
CACCTGGAGGCCACAGCACACCGTCTCAACAAAGAGAACCCGTCAATCCG
TGCCTTCGTGAGCGTGAATAAAGGCCTGGTAACCCGCGCAAGCGCCGTAA
TCCCGGTAATCCCTCTGTATCTCGCCAGCTTGTTCAAAGTAATGAAAGAG
AAGGGCAATCATGAAGGTTGTATTGAACAGATCACGCGTCTGTACGCCGA
GCGCCTGTACCGTAAAGATGGTACAATTCCAGTTGATGAGGAAAATCGCA
TTCGCATTGATGATTGGGAGTTAGAAGAAGACGTCCAGAAAGCGGTATCC
GCGTTGATGGAGAAAGTCACGGGTGAAAACGCAGAATCTCTCACTGACTT
AGCGGGGTACCGCCATGATTTCTTAGCTAGTAACGGCTTTGATGTAGAAG
GTATTAATTATGAAGCGGAAGTTGAACGCTTCGACCGTATCTGA
ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGAAAAAAT
TGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTTACGCCAGG
TGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAAAAGAGACC
GTCCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTACTTTCTTCGCCC
TGGCGATAGTAAGAAGCCGATTATTTATGATGTCGAAACGCTGCGTGACG
GTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCG
ATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACA
TCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAA
tesB CGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGAT
SEQ ID NO: 10 AAATTCATCTGCGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAA
CCCACTGAAAGGTCACGTCGCAGAACCACATCGTCAGGTGTGGATCCGCG
CAAATGGTAGCGTGCCGGATGACCTGCGCGTTCATCAGTATCTGCTCGGT
TACGCTTCTGATCTTAACTTCCTGCCGGTAGCTCTACAGCCGCACGGCAT
CGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGT
GGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGGAG
AGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATAC
CCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTA
ATCACAATTAA

[0239] Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 3.
Table 3. Exemplary Polypeptide Sequences for Butyrate Production Description Sequence Bcd2 MDLNS KKYQMLKELYVSFAENEVKPLATELDEEER
SEQ ID NO: 11 FPYETVEKMAKAGMMGIPYPKEYGGEGGDTVGYIM
AVEELSRVC GTTGVILS AHTSLGS WPIYQYGNEEQK
QKFLRPLAS GE KLGAFGLTEPNAGTD AS GQQTTAVL
DGDEYILNGS KIFITNAIAGDIYVVMAMTD KS KGNK
GIS AFIVEKGTPGFS FGVKEKKMGIRGS AT S ELIFEDC
RIPKENLLGKEGQGFKIAMS TLDGGRIGIAAQALGLA
QGALDETVKYVKERVQFGRPLS KFQNTQFQLADME
VKVQAARHLVYQAAINKDLGKPYGVEAAMAKLFA
AETAMEVTTKAVQLHGGYGYTRDYPVERMMRDAK
ITEIYEGTSEVQRMVIS GKLLK
etfB 3 MNIVVCIKQVPDTTEVKLDPNTGTLIRDGVPSIINPDD
SEQ ID NO: 12 KAGLEEAIKLKEEMGAHVTVITMGPPQADMALKEA
LAMGADRGILLTDRAFAGADTWATS S ALAGALKNI
DFDIIIAGRQAIDGDTAQVGPQIAEHLNLPSITYAEEIK
TEGEYVLVKRQFEDCCHDLKVKMPCLITTLKDMNT
PRYMKVGRIYDAFENDVVETWTVKDIEVDPSNLGL
KGS PT S VFKS FT KS VKPAGTIYNEDAKTS AGIIIDKLK
EKYII
etfA3 MGNVLVVIEQRENVIQTVSLELLGKATEIAKDYDTK
SEQ ID NO: 13 VS ALLLGS KVEGLIDTLAHYGADEVIVVDDEALAVY
TTEPYT KAAYE AI KAADPIVVLFGAT S IGRDLAPRVS
ARIHTGLTADCTGLAVAEDTKLLLMTRPAFGGNIMA
TIVCKDFRPQMS TVRPGVMKKNEPDETKEAVINRFK
VEFND AD KLVQVVQVI KE AKKQV KIEDAKILVS AGR
GMGGKENLDILYELAEIIGGEVS GS RATIDAGWLD K
ARQVGQT GKTVRPDLYIAC GIS GAIQHIAGMEDAEFI
VAIN KNPE APIFKYADVGIVGD VH KVLPELIS QLS VA
KEKGEVLAN
Ter MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRIT
SEQ ID NO: 14 AEVKAGAKAPKNVLVLGCSNGYGLASRITAAFGYG
AATIGVS FE KAGS ET KYGTPGWYNNLAFDEAAKRE
GLYS VTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYSL
AS PVRTDPDT GIMH KS VLKPFG KTFT G KTVD PFT GEL
KEIS AEPANDEEAAATVKVMGGEDWERWIKQLS KE
GLLEEGCITLAYS YIGPEATQALYRKGTIGKAKEHLE
AT AHRLN KENPS IRAFVS VNKGLVTRAS AVIPVIPLY
LAS LFKVM KE KGNHE GCIE QITRLYAERLYRKD GTIP
VDEENRIRIDDWELEEDVQKAVS ALMEKVT GENAES
LTDLAGYRHDFLASNGFDVEGINYEAEVERFDRI
ThiA MREVVIAS AARTAVGS FGGAFKS VS AVELGVTAAK
SEQ ID NO: 15 EAIKRANITPDMIDESLLGGVLTAGLGQNIARQIALG

AGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDADI
MLVGGAENMSMSPYLVPSARYGARMGDAAFVDSM
IKDGLSDIFNNYHMGITAENIAEQWNITREEQDELAL
AS QNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDK
DEYIKPGTTMEKLAKLRPAFKKDGTVTAGNASGIND
GAAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKI
MGYGPVPATKKALEAANMTIEDIDLVEANEAFAAQ
SVAVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILT
TLLYEMKRRDAKTGLATLCIGGGMGTTLIVKR
Hbd MKLAVIGSGTMGSGIVQTFASCGHDVCLKSRTQGAI
SEQ ID NO: 16 DKCLALLDKNLT KLVT KGKMDEAT KAEILS HVS S TT
NYEDLKDMDLIIEASVEDMNIKKDVFKLLDELCKED
TILATNTSSLSITEIASSTKRPDKVIGMHFFNPVPMMK
LVEVISGQLTSKVTFDTVFELSKSINKVPVDVSESPGF
VVNRILIPMINEAVGIYADGVASKEEIDEAMKLGAN
HPMGPLALGDLIGLDVVLAIMNVLYTEFGDTKYRPH
PLLAKMVRANQLGRKTKIGFYDYNK
Crt2 MS TSDVKVYENVAVEVDGNICTVKMNRPKALNAIN
SEQ ID NO: 17 SKTLEELYEVFVDINNDETIDVVILTGEGKAFVAGAD
IAYMKDLDAVAAKDFSILGAKAFGEIENSKKVVIAA
VNGFALGGGCELAMACDIRIASAKAKFGQPEVTLGI
TPGYGGTQRLTRLVGMAKAKELIFTGQVIKADEAEK
IGLVNRVVEPDILIEEVEKLAKIIAKNAQLAVRYSKE
AIQLGAQTDINTGIDIESNLFGLCFSTKDQKEGMSAF
VEKREANFIKG
Pbt MRSFEEVIKFAKERGPKTISVACCQDKEVLMAVEMA
SEQ ID NO: 18 RKEKIANAILVGDIEKTKEIAKSIDMDIENYELIDIKD
LAEASLKSVELVSQGKADMVMKGLVDTSIILKAVLN
KEVGLRTGNVLSHVAVFDVEGYDRLFFVTDAAMNL
APDTNTKKQIIENACTVAHSLDISEPKVAAICAKEKV
NPKMKDTVEAKELEEMYERGEIKGCMVGGPFAIDN
AVSLEAAKHKGINHPVAGRADILLAPDIEGGNILYKA
LVFFS KS KNAGVIVGAKAPIILTSRADSEETKLNSIAL
GVLMAAKA
Buk MS KIFKILTINPGSTSTKIAVFDNEDLVFEKTLRHS SE
SEQ ID NO: 19 EIGKYEKVSDQFEFRKQVIEEALKEGGVKTSELDAV
VGRGGLLKPIKGGTYS VS AAMIEDLKVGVLGEHASN
LGGIIAKQIGEEVNVPS YIVDPVVVDELEDVARIS GM
PEISRASVVHALNQKAIARRYAREINKKYEDINLIVA
HMGGGVSVGAHKNGKIVDVANALDGEGPFSPERSG
GLPVGALVKMCFSGKYTQDEIKKKIKGNGGLVAYL
NTNDAREVEERIEAGDEKAKLVYEAMAYQISKEIGA
SAAVLKGDVKAILLTGGIAYSKMFTEMIADRVKFIA
DVKVYPGEDEMIALAQGGLRVLTGEEEAQVYDN

TesB MS QALKNLLTLINLEKIEEGLFRGQS EDLGLRQVFG
SEQ ID NO: 20 GQV VGQALYAAKETV PEERLVHSFES YFLRYGDSKK
PIINDVETLRDGNS FS ARR VA AIQNGKPIFYMTASFQ
APEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLLPP
KD K FICD IZPLE RPV EFfi NPLKGH AE PH RQVW1 RANGS VPDDLR VIIQYLLGY ASDLNFLPV ALQPII GIG
FLEPGIQIATIDEISMVVETIRPFN LNEWLLYS VESTSAS
S ARG FV RGEFYTQ DGVLVASTVQEG VM
[0240] The gene products of the bcd2, e03, and etf733 genes in Clostridium difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant. In some embodiments, because the genetically engineered bacteria of the invention are designed to produce butyrate in a microaerobic or oxygen-limited environment, e.g., the mammalian gut, oxygen dependence could have a negative effect on butyrate production in the gut. It has been shown that a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) can functionally replace this three-gene complex in an oxygen-independent manner. In some embodiments, the genetically engineered bacteria comprise a ter gene, e.g., from Treponema denticola, which can functionally replace all three of the bcd2, etf733, and etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the genetically engineered bacteria comprise thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose..
[0241] In some embodiments, the genetically engineered bacteria of the invention comprise thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile; ter, e.g., from Treponema denticola; one or more of bcd2, etf733, and etfA3, e.g., from Peptoclostridium difficile; and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0242] The gene products of pbt and buk convert butyrylCoA to Butyrate. In some embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA. In one embodiment, the genetically engineered bacteria comprise bcd2, etf733, e03, thiAl, hbd, and crt2, e.g., from Peptoclostridium chfficile, and tesB from E. Coli and produce butyrate in low-oxygen conditions, in the presence of molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
In one embodiment, the genetically engineered bacteria comprise ter gene (encoding trans-2-enoynl-CoA reductase) e.g., from Treponema denticola, thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium chfficile, and tesB from E. Coli , and produce butyrate in low-oxygen conditions,in the presence of specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0243] In some embodiments, the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis. In some embodiments, local butyrate production reduces gut inflammation, a symptom of IBD and other gut related disorders.
[0244] In one embodiment, the bcd2 gene has at least about 80% identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about 85%
identity with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 90%
identity with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 95%
identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. Accordingly, in one embodiment, the bcd2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 1. In yet another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 1.
[0245] In one embodiment, the eff133 gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the eff133 gene has at least about 85%
identity with SEQ ID NO: 2. In one embodiment, the etf733 gene has at least about 90%
identity with SEQ ID NO: 2. In one embodiment, the etf733 gene has at least about 95%
identity with SEQ ID NO: 2. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the effB3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. In another embodiment, the etf733 gene comprises the sequence of SEQ ID NO: 2. In yet another embodiment the effB3 gene consists of the sequence of SEQ ID NO: 2.
[0246] In one embodiment, the effA3 gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the effA3 gene has at least about 85%
identity with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 90%
identity with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 95%
identity with SEQ ID NO: 3. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the effA3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 3. In yet another embodiment the effA3 gene consists of the sequence of SEQ ID NO: 3.
[0247] In one embodiment, the thiAl gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the thiAl gene has at least about 85%
identity with SEQ ID NO: 4. In one embodiment, the thiAl gene has at least about 90%
identity with SEQ ID NO: 4. In one embodiment, the thiAl gene has at least about 95%
identity with SEQ ID NO: 4. In another embodiment, the thiAl gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the thiAl gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In another embodiment, the thiAl gene comprises the sequence of SEQ ID NO: 4. In yet another embodiment the thiAl gene consists of the sequence of SEQ ID NO: 4.
[0248] In one embodiment, the hbd gene has at least about 80% identity with SEQ
ID NO: 5. In another embodiment, the hbd gene has at least about 85% identity with SEQ
ID NO: 5. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID
NO: 5. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID
NO: 5. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99%
identity with SEQ ID NO: 5. Accordingly, in one embodiment, the hbd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 5.
[0249] In one embodiment, the crt2 gene has at least about 80% identity with SEQ
ID NO: 6. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the crt2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 6. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 6.
[0250] In one embodiment, the pbt gene has at least about 80% identity with SEQ
ID NO: 7. In another embodiment, the pbt gene has at least about 85% identity with SEQ
ID NO: 7. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID
NO: 7. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID
NO: 7. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99%
identity with SEQ ID NO: 7. Accordingly, in one embodiment, the pbt gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 7.
[0251] In one embodiment, the buk gene has at least about 80% identity with SEQ
ID NO: 8. In another embodiment, the buk gene has at least about 85% identity with SEQ
ID NO: 8. In one embodiment, the buk gene has at least about 90% identity with SEQ ID
NO: 8. In one embodiment, the buk gene has at least about 95% identity with SEQ ID
NO: 8. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99%
identity with SEQ ID NO: 8. Accordingly, in one embodiment, the buk gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 8. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 8.
[0252] In one embodiment, the ter gene has at least about 80% identity with SEQ
ID NO: 9. In another embodiment, the ter gene has at least about 85% identity with SEQ
ID NO: 9. In one embodiment, the ter gene has at least about 90% identity with SEQ ID
NO: 9. In one embodiment, the ter gene has at least about 95% identity with SEQ ID
NO: 9. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99%
identity with SEQ ID NO: 9. Accordingly, in one embodiment, the ter gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 9.
[0253] In one embodiment, the tesB gene has at least about 80% identity with SEQ
ID NO: 10. In another embodiment, the tesB gene has at least about 85%
identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90%
identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95%
identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the tesB
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 10.
[0254] In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%
identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 11 through SEQ ID
NO: 20.
In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95%
identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID
NO: 11 through SEQ ID NO: 20. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID
NO: 11 through SEQ ID NO: 20. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 11 through SEQ ID
NO: 20.
[0255] In some embodiments, one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Peptoclostridicum difficile butyrate biosynthesis gene. The butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.
[0256] To improve acetate production, while maintaining high levels of butyrate production, one or more targeted deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of such competing metabolic arms are frdA
(converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more butyrate-producing cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE genes.
[0257] In some embodiments, the genetically engineered bacteria comprise one or more butyrate producing cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE
gene.
[0258] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
genes.
[0259] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0260] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB
and further comprise a mutation and/or deletion in the endogenous adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB
and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB
gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0261] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce 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 acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0262] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0263] In certain situations, the need may arise to prevent and/or reduce acetate production of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of butyrate production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for butyrate production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for butyrate synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE
genes.
[0264] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE
genes.
[0265] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA
gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA
gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and ldhA genes.
[0266] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0267] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB
and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and ldhA genes.
[0268] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB
butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA
and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0269] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0270] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0271] In some embodiments, the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production. In some embodiments, the local production of butyrate reduces food intake and ameliorates improves gut barrier function and reduces inflammation. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0272] In one embodiment, the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the butyrate gene cassette in nature.
[0273] In some embodiments, the butyrate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the butyrate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.
[0274] The butyrate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the butyrate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the butyrate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located on a plasmid in the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.
[0275] In some embodiments, the butyrate gene cassette is expressed on a low-copy plasmid. In some embodiments, the butyrate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of butyrate.
Propionate [0276] In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing an anti-inflammatory or gut barrier enhancer molecule, e.g., propionate, that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.
[0277] In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions. The genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 4, Table 5, Table 6, Table 7). Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. In some embodiments, the genetically engineered bacteria of the invention comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, e0, acrB, and acrC. In some embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the AcuI from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of AcuI in E coli, yhdH is used. This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and 1pd, and optionally further comprise tesB. In another embodiment, the propionate gene cassette comprises the genes of the Sleepting Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm converts succinyl CoA to L-methylmalonylCoA, ygfG
converts L-methylmalonylCoA into PropionylCoA, and ygfH converts propionylCoA
into propionate and succinate into succinylCoA.
[0278] This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134).

There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD 18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.
[0279] The genes may be codon-optimized, and translational and transcriptional elements may be added. Table 4-6 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 7 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes.
Table 4. Propionate Cassette Sequences (Acrylate Pathway) Gene sequence Description ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAAC
TGATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGT
AACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTT
CCTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCG
GCAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGC
GCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGA
CCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGA
GGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGA
TATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCG
GCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGAT
pct ATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGC
SEQ ID NO: 21 AGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTG
ATTCGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTTGA
AAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGG
TGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTC
GTCAAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGG
GTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCAT
CAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGA
GCACCGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCG
CGAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAA
AGACGTTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGG

Gene sequence Description CCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTC
ACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGT
TCGCTTTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGG
GCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTT
ACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTT
AGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAA
CATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTA
CAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGAT
TATCGTTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTG
GAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGC
AACAAGTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAA
GAAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCT
GCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTG
ATCGTGACGCAAACGGGCAGATTAAACTGATGGACGCGGCGTT
ATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTAA
ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCAT
ACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGC
CCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG
AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACT
CATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGC
TGGAAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCC
TACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGC
CGCCATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTG
CTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATA
TCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGA
ACTCGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATAC
CATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGT
TCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGT
TCGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCG
lcdA =TAGCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATAC
SEQ ID NO: 22 AAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGC
GTTAATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCT
TTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGG
TATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGG
AAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCT
ATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGC
CCTTTGGGACCTGCACTATGACGCTAACGACGAATCTATGCACT
CTATGGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGC
AGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCA
GGTGGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGA
TGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAA
GAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATC
CTCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCC
TGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAA

Gene sequence Description ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC
CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACA
GGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAG
AAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGC
GCCCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGC
TTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCG
AGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCG
TGCGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTC
CCCAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAG
CGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCA
CAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCT
GGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGC
lcdB GTGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGAC
SEQ ID NO: 23 GCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCT
TAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAG
ATTAAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTG
TAGTGACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATC
TTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCA
GGAAAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGC
GGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGG
CTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGT
TAATTAACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTT
GCAATGATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGT
AATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATG
ATTGAGGTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAAC
CCGTCTGCAGTCATTCGTCGAAATGCTTTAA
ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGC
GGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTG
TCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC
AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTA
CACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGG
ATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTAT
TTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCA
AGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAG
lcdC CAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTT
SEQ ID NO: 24 CCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAA
TGGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCA
AGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATT
GAGCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATC
TGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGT
TTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATG
CAGGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGT
TATCGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAG
CGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTA
etfA ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGG
SEQ ID NO: 25 GTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATTT

Gene sequence Description CGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGA
AGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAA
TCGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGT
GTGCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATG
CCAAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGAGGT
AATTTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGC
GTTGTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACG
CACCTGGATATTGATATGAATAAATATGTGGACTTTCTTAGCAC
CAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAG
ATACAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTG
ATGGCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCAC
AGTGCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAG
ATGGCGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTC
GGATGAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAA
ACGAAAAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTC
AGTTGGTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGCAC
TGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGC
GGCTCGCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCA
TCAGGTTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTAC
GTGGCGCTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGAT
GCAAGACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACG
GCGCCTATCTTCGACTGCGCCGATTATGGCATCACCGGTGATTT
ATTTAAAATCGTACCGATGATGATCGACGCGATCAAAGAGGGT
AAAAACGCATGA
ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGG
CAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAA
TGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAG
GCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGC
TTACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATT
ATTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGGCCCGTGA
ATTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCG
CGGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTT
TGCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCC
acrB
TCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCG
SEQ ID NO: 26 CAGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATG
TTGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCT
GATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGA
CTCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC
TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGAT
ACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTT
TACGCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCACCG
ATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAA
ACATGTCATCTAA

Gene sequence Description ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGG
ACTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCG
CGTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGA
TTGATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATG
GCGGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGG
TGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAG
AATTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATT
CATCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGA
ACAGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAA
CTGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATG
CCGGCGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAA
CGAGTACGTGCTGAATGGCACCAAATGCTTTATCAGCGGGGGCG
GGCGCGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAA
acrC AAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGA
SEQ ID NO: 27 CCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGAT
CGCAGGTTCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCG
TTCCGGCTGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAA
ATTGCTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGC
TCAAGCAATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTG
AAGTACGTTCACGAGCGCATTCAATTTGGTAAACCGATCGCGAA
TCTGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAA
CCGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAA
GACGCGGGTAAACCGTTCACAAAGGAATCTGCTATGTGCAAGCT
GAACGCCTCCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGC
AGATTCACGGGGGTTACGGTTATATGAAAGATTATCCGTTAGAG
CGTATGTATCGCGATGCTAAGATTACGGAAATTTACGAGGGGAC
ATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAA
CGCTAA
ATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGGCAAATGCAG
AACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCCAGG
CAGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCAC
CAACCACCTGGTGGCGATGATTGAAAAAACCATTAGCGGCCAG
GATGCTTTACCCAATATCAGCGATGCCGAACGTATTTTTGCCGA
ACTTTTGACGGGACTCGCCGCCGCCCAGCCGGGGTTCCCGCTGG
CGCAATTGAAAACTTTCGTCGATCAGGAATTTGCCCAAATAAAA
CATGTCCTGCATGGCATTAGTTTGTTGGGGCAGTGCCCGGATAG
thrgbr CATCAACGCTGCGCTGATTTGCCGTGGCGAGAAAATGTCGATCG
SEQ ID NO: 28 CCATTATGGCCGGCGTATTAGAAGCGCGCGGTCACAACGTTACT
GTTATCGATCCGGTCGAAAAACTGCTGGCAGTGGGGCATTACCT
CGAATCTACCGTCGATATTGCTGAGTCCACCCGCCGTATTGCGG
CAAGCCGCATTCCGGCTGATCACATGGTGCTGATGGCAGGTTTC
ACCGCCGGTAATGAAAAAGGCGAACTGGTGGTGCTTGGACGCA
ACGGTTCCGACTACTCTGCTGCGGTGCTGGCTGCCTGTTTACGC
GCCGATTGTTGCGAGATTTGGACGGACGTTGACGGGGTCTATAC
CTGCGACCCGCGTCAGGTGCCCGATGCGAGGTTGTTGAAGTCGA
TGTCCTACCAGGAAGCGATGGAGCTTTCCTACTTCGGCGCTAAA

Gene sequence Description GTTCTTCACCCCCGCACCATTACCCCCATCGCCCAGTTCCAGATC
CCTTGCCTGATTAAAAATACCGGAAATCCTCAAGCACCAGGTAC
GCTCATTGGTGCCAGCCGTGATGAAGACGAATTACCGGTCAAGG
GCATTTCCAATCTGAATAACATGGCAATGTTCAGCGTTTCTGGT
CCGGGGATGAAAGGGATGGTCGGCATGGCGGCGCGCGTCTTTG
CAGCGATGTCACGCGCCCGTATTTCCGTGGTGCTGATTACGCAA
TCATCTTCCGAATACAGCATCAGTTTCTGCGTTCCACAAAGCGA
CTGTGTGCGAGCTGAACGGGCAATGCAGGAAGAGTTCTACCTG
GAACTGAAAGAAGGCTTACTGGAGCCGCTGGCAGTGACGGAAC
GGCTGGCCATTATCTCGGTGGTAGGTGATGGTATGCGCACCTTG
CGTGGGATCTCGGCGAAATTCTTTGCCGCACTGGCCCGCGCCAA
TATCAACATTGTCGCCATTGCTCAGAGATCTTCTGAACGCTCAA
TCTCTGTCGTGGTAAATAACGATGATGCGACCACTGGCGTGCGC
GTTACTCATCAGATGCTGTTCAATACCGATCAGGTTATCGAAGT
GTTTGTGATTGGCGTCGGTGGCGTTGGCGGTGCGCTGCTGGAGC
AACTGAAGCGTCAGCAAAGCTGGCTGAAGAATAAACATATCGA
CTTACGTGTCTGCGGTGTTGCCAACTCGAAGGCTCTGCTCACCA
ATGTACATGGCCTTAATCTGGAAAACTGGCAGGAAGAACTGGC
GCAAGCCAAAGAGCCGTTTAATCTCGGGCGCTTAATTCGCCTCG
TGAAAGAATATCATCTGCTGAACCCGGTCATTGTTGACTGCACT
TCCAGCCAGGCAGTGGCGGATCAATATGCCGACTTCCTGCGCGA
AGGTTTCCACGTTGTCACGCCGAACAAAAAGGCCAACACCTCGT
CGATGGATTACTACCATCAGTTGCGTTATGCGGCGGAAAAATCG
CGGCGTAAATTCCTCTATGACACCAACGTTGGGGCTGGATTACC
GGTTATTGAGAACCTGCAAAATCTGCTCAATGCAGGTGATGAAT
TGATGAAGTTCTCCGGCATTCTTTCTGGTTCGCTTTCTTATATCTT
CGGCAAGTTAGACGAAGGCATGAGTTTCTCCGAGGCGACCACG
CTGGCGCGGGAAATGGGTTATACCGAACCGGACCCGCGAGATG
ATCTTTCTGGTATGGATGTGGCGCGTAAACTATTGATTCTCGCTC
GTGAAACGGGACGTGAACTGGAGCTGGCGGATATTGAAATTGA
ACCTGTGCTGCCCGCAGAGTTTAACGCCGAGGGTGATGTTGCCG
CTTTTATGGCGAATCTGTCACAACTCGACGATCTCTTTGCCGCGC
GCGTGGCGAAGGCCCGTGATGAAGGAAAAGTTTTGCGCTATGTT
GGCAATATTGATGAAGATGGCGTCTGCCGCGTGAAGATTGCCGA
AGTGGATGGTAATGATCCGCTGTTCAAAGTGAAAAATGGCGAA
AACGCCCTGGCCTTCTATAGCCACTATTATCAGCCGCTGCCGTT
GGTACTGCGCGGATATGGTGCGGGCAATGACGTTACAGCTGCCG
GTGTCTTTGCTGATCTGCTACGTACCCTCTCATGGAAGTTAGGA
GTCTGA

Gene sequence Description ATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGT
CGGGTTTGATGTGCTCGGGGCGGCGGTGACACCTGTTGATGGTG
CATTGCTCGGAGATGTAGTCACGGTTGAGGCGGCAGAGACATTC
AGTCTCAACAACCTCGGACGCTTTGCCGATAAGCTGCCGTCAGA
ACCACGGGAAAATATCGTTTATCAGTGCTGGGAGCGTTTTTGCC
AGGAACTGGGTAAGCAAATTCCAGTGGCGATGACCCTGGAAAA
GAATATGCCGATCGGTTCGGGCTTAGGCTCCAGTGCCTGTTCGG
TGGTCGCGGCGCTGATGGCGATGAATGAACACTGCGGCAAGCC
GCTTAATGACACTCGTTTGCTGGCTTTGATGGGCGAGCTGGAAG
GCCGTATCTCCGGCAGCATTCATTACGACAACGTGGCACCGTGT
thrB TTTCTCGGTGGTATGCAGTTGATGATCGAAGAAAACGACATCAT
SEQ ID NO: 29 CAGCCAGCAAGTGCCAGGGTTTGATGAGTGGCTGTGGGTGCTGG
CGTATCCGGGGATTAAAGTCTCGACGGCAGAAGCCAGGGCTATT
TTACCGGCGCAGTATCGCCGCCAGGATTGCATTGCGCACGGGCG
ACATCTGGCAGGCTTCATTCACGCCTGCTATTCCCGTCAGCCTG
AGCTTGCCGCGAAGCTGATGAAAGATGTTATCGCTGAACCCTAC
CGTGAACGGTTACTGCCAGGCTTCCGGCAGGCGCGGCAGGCGG
TCGCGGAAATCGGCGCGGTAGCGAGCGGTATCTCCGGCTCCGGC
CCGACCTTGTTCGCTCTGTGTGACAAGCCGGAAACCGCCCAGCG
CGTTGCCGACTGGTTGGGTAAGAACTACCTGCAAAATCAGGAA
GGTTTTGTTCATATTTGCCGGCTGGATACGGCGGGCGCACGAGT
ACTGGAAAACTAA
ATGAAACTCTACAATCTGAAAGATCACAACGAGCAGGTCAGCTT
TGCGCAAGCCGTAACCCAGGGGTTGGGCAAAAATCAGGGGCTG
TTTTTTCCGCACGACCTGCCGGAATTCAGCCTGACTGAAATTGA
TGAGATGCTGAAGCTGGATTTTGTCACCCGCAGTGCGAAGATCC
TCTCGGCGTTTATTGGTGATGAAATCCCACAGGAAATCCTGGAA
GAGCGCGTGCGCGCGGCGTTTGCCTTCCCGGCTCCGGTCGCCAA
TGTTGAAAGCGATGTCGGTTGTCTGGAATTGTTCCACGGGCCAA
CGCTGGCATTTAAAGATTTCGGCGGTCGCTTTATGGCACAAATG
CTGACCCATATTGCGGGTGATAAGCCAGTGACCATTCTGACCGC
GACCTCCGGTGATACCGGAGCGGCAGTGGCTCATGCTTTCTACG
GTTTACCGAATGTGAAAGTGGTTATCCTCTATCCACGAGGCAAA
thrC ATCAGTCCACTGCAAGAAAAACTGTTCTGTACATTGGGCGGCAA
SEQ ID NO: 30 TATCGAAACTGTTGCCATCGACGGCGATTTCGATGCCTGTCAGG
CGCTGGTGAAGCAGGCGTTTGATGATGAAGAACTGAAAGTGGC
GCTAGGGTTAAACTCGGCTAACTCGATTAACATCAGCCGTTTGC
TGGCGCAGATTTGCTACTACTTTGAAGCTGTTGCGCAGCTGCCG
CAGGAGACGCGCAACCAGCTGGTTGTCTCGGTGCCAAGCGGAA
ACTTCGGCGATTTGACGGCGGGTCTGCTGGCGAAGTCACTCGGT
CTGCCGGTGAAACGTTTTATTGCTGCGACCAACGTGAACGATAC
CGTGCCACGTTTCCTGCACGACGGTCAGTGGTCACCCAAAGCGA
CTCAGGCGACGTTATCCAACGCGATGGACGTGAGTCAGCCGAA
CAACTGGCCGCGTGTGGAAGAGTTGTTCCGCCGCAAAATCTGGC
AACTGAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCAC
GCAACAGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCG

Gene sequence Description GAGCCGCACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTT
GAATCCAGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGG
CGAAATTTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTT
GGATCTGCCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGC
TTTCACATAATCTGCCCGCCGATTTTGCTGCGTTGCGTAAATTGA
TGATGAATCATCAGTAA
ATGAGTGAAACATACGTGTCTGAGAAAAGTCCAGGAGTGATGG
CTAGCGGAGCGGAGCTGATTCGTGCCGCCGACATTCAAACGGC
GCAGGCACGAATTTCCTCCGTCATTGCACCAACTCCATTGCAGT
ATTGCCCTCGTCTTTCTGAGGAAACCGGAGCGGAAATCTACCTT
AAGCGTGAGGATCTGCAGGATGTTCGTTCCTACAAGATCCGCGG
TGCGCTGAACTCTGGAGCGCAGCTCACCCAAGAGCAGCGCGAT
GCAGGTATCGTTGCCGCATCTGCAGGTAACCATGCCCAGGGCGT
GGCCTATGTGTGCAAGTCCTTGGGCGTTCAGGGACGCATCTATG
TTCCTGTGCAGACTCCAAAGCAAAAGCGTGACCGCATCATGGTT
CACGGCGGAGAGTTTGTCTCCTTGGTGGTCACTGGCAATAACTT
CGACGAAGCATCGGCTGCAGCGCATGAAGATGCAGAGCGCACC
GGCGCAACGCTGATCGAGCCTTTCGATGCTCGCAACACCGTCAT
CGGTCAGGGCACCGTGGCTGCTGAGATCTTGTCGCAGCTGACTT
CCATGGGCAAGAGTGCAGATCACGTGATGGTTCCAGTCGGCGGT
i/vAfbr GGCGGACTTCTTGCAGGTGTGGTCAGCTACATGGCTGATATGGC
SEQ ID NO: 31 ACCTCGCACTGCGATCGTTGGTATCGAACCAGCGGGAGCAGCAT
CCATGCAGGCTGCATTGCACAATGGTGGACCAATCACTTTGGAG
ACTGTTGATCCCTTTGTGGACGGCGCAGCAGTCAAACGTGTCGG
AGATCTCAACTACACCATCGTGGAGAAGAACCAGGGTCGCGTG
CACATGATGAGCGCGACCGAGGGCGCTGTGTGTACTGAGATGCT
CGATCTTTACCAAAACGAAGGCATCATCGCGGAGCCTGCTGGCG
CGCTGTCTATCGCTGGGTTGAAGGAAATGTCCTTTGCACCTGGT
TCTGCAGTGGTGTGCATCATCTCTGGTGGCAACAACGATGTGCT
GCGTTATGCGGAAATCGCTGAGCGCTCCTTGGTGCACCGCGGTT
TGAAGCACTACTTCTTGGTGAACTTCCCGCAAAAGCCTGGTCAG
TTGCGTCACTTCCTGGAAGATATCCTGGGACCGGATGATGACAT
CACGCTGTTTGAGTACCTCAAGCGCAACAACCGTGAGACCGGTA
CTGCGTTGGTGGGTATTCACTTGAGTGAAGCATCAGGATTGGAT
TCTTTGCTGGAACGTATGGAGGAATCGGCAATTGATTCCCGTCG
CCTCGAGCCGGGCACCCCTGAGTACGAATACTTGACCTAA
ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCG
CGACTGGCTCCAGGCGATCGAATCGGTCATCCGTGAAGAAGGT
GTTGAGCGTGCTCAGTATCTGATCGACCAACTGCTTGCTGAAGC
CCGCAAAGGCGGTGTAAACGTAGCCGCAGGCACAGGTATCAGC
aceE AACTACATCAACACCATCCCCGTTGAAGAACAACCGGAGTATCC
SEQ ID NO: 32 GGGTAATCTGGAACTGGAACGCCGTATTCGTTCAGCTATCCGCT
GGAACGCCATCATGACGGTGCTGCGTGCGTCGAAAAAAGACCT
CGAACTGGGCGGCCATATGGCGTCCTTCCAGTCTTCCGCAACCA
TTTATGATGTGTGCTTTAACCACTTCTTCCGTGCACGCAACGAGC
AGGATGGCGGCGACCTGGTTTACTTCCAGGGCCACATCTCCCCG

Gene sequence Description GGCGTGTACGCTCGTGCTTTCCTGGAAGGTCGTCTGACTCAGGA
GCAGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCT
CTTCCTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTC
CCGACCGTATCTATGGGTCTGGGTCCGATTGGTGCTATTTACCA
GGCTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATA
CCTCTAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATG
GACGAACCGGAATCCAAAGGTGCGATCACCATCGCTACCCGTG
AAAAACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAG
CGTCTTGACGGCCCGGTCACCGGTAACGGCAAGATCATCAACGA
ACTGGAAGGCATCTTCGAAGGTGCTGGCTGGAACGTGATCAAA
GTGATGTGGGGTAGCCGTTGGGATGAACTGCTGCGTAAGGATAC
CAGCGGTAAACTGATCCAGCTGATGAACGAAACCGTTGACGGC
GACTACCAGACCTTCAAATCGAAAGATGGTGCGTACGTTCGTGA
ACACTTCTTCGGTAAATATCCTGAAACCGCAGCACTGGTTGCAG
ACTGGACTGACGAGCAGATCTGGGCACTGAACCGTGGTGGTCA
CGATCCGAAGAAAATCTACGCTGCATTCAAGAAAGCGCAGGAA
ACCAAAGGCAAAGCGACAGTAATCCTTGCTCATACCATTAAAG
GTTACGGCATGGGCGACGCGGCTGAAGGTAAAAACATCGCGCA
CCAGGTTAAGAAAATGAACATGGACGGTGTGCGTCATATCCGC
GACCGTTTCAATGTGCCGGTGTCTGATGCAGATATCGAAAAACT
GCCGTACATCACCTTCCCGGAAGGTTCTGAAGAGCATACCTATC
TGCACGCTCAGCGTCAGAAACTGCACGGTTATCTGCCAAGCCGT
CAGCCGAACTTCACCGAGAAGCTTGAGCTGCCGAGCCTGCAAG
ACTTCGGCGCGCTGTTGGAAGAGCAGAGCAAAGAGATCTCTAC
CACTATCGCTTTCGTTCGTGCTCTGAACGTGATGCTGAAGAACA
AGTCGATCAAAGATCGTCTGGTACCGATCATCGCCGACGAAGCG
CGTACTTTCGGTATGGAAGGTCTGTTCCGTCAGATTGGTATTTAC
AGCCCGAACGGTCAGCAGTACACCCCGCAGGACCGCGAGCAGG
TTGCTTACTATAAAGAAGACGAGAAAGGTCAGATTCTGCAGGA
AGGGATCAACGAGCTGGGCGCAGGTTGTTCCTGGCTGGCAGCG
GCGACCTCTTACAGCACCAACAATCTGCCGATGATCCCGTTCTA
CATCTATTACTCGATGTTCGGCTTCCAGCGTATTGGCGATCTGTG
CTGGGCGGCTGGCGACCAGCAAGCGCGTGGCTTCCTGATCGGCG
GTACTTCCGGTCGTACCACCCTGAACGGCGAAGGTCTGCAGCAC
GAAGATGGTCACAGCCACATTCAGTCGCTGACTATCCCGAACTG
TATCTCTTACGACCCGGCTTACGCTTACGAAGTTGCTGTCATCAT
GCATGACGGTCTGGAGCGTATGTACGGTGAAAAACAAGAGAAC
GTTTACTACTACATCACTACGCTGAACGAAAACTACCACATGCC
GGCAATGCCGGAAGGTGCTGAGGAAGGTATCCGTAAAGGTATC
TACAAACTCGAAACTATTGAAGGTAGCAAAGGTAAAGTTCAGC
TGCTCGGCTCCGGTTCTATCCTGCGTCACGTCCGTGAAGCAGCT
GAGATCCTGGCGAAAGATTACGGCGTAGGTTCTGACGTTTATAG
CGTGACCTCCTTCACCGAGCTGGCGCGTGATGGTCAGGATTGTG
AACGCTGGAACATGCTGCACCCGCTGGAAACTCCGCGCGTTCCG
TATATCGCTCAGGTGATGAACGACGCTCCGGCAGTGGCATCTAC
CGACTATATGAAACTGTTCGCTGAGCAGGTCCGTACTTACGTAC

Gene sequence Description CGGCTGACGACTACCGCGTACTGGGTACTGATGGCTTCGGTCGT
TCCGACAGCCGTGAGAACCTGCGTCACCACTTCGAAGTTGATGC
TTCTTATGTCGTGGTTGCGGCGCTGGGCGAACTGGCTAAACGTG
GCGAAATCGATAAGAAAGTGGTTGCTGACGCAATCGCCAAATT
CAACATCGATGCAGATAAAGTTAACCCGCGTCTGGCGTAA
ATGGCTATCGAAATCAAAGTACCGGACATCGGGGCTGATGAAG
TTGAAATCACCGAGATCCTGGTCAAAGTGGGCGACAAAGTTGA
AGCCGAACAGTCGCTGATCACCGTAGAAGGCGACAAAGCCTCT
ATGGAAGTTCCGTCTCCGCAGGCGGGTATCGTTAAAGAGATCAA
AGTCTCTGTTGGCGATAAAACCCAGACCGGCGCACTGATTATGA
TTTTCGATTCCGCCGACGGTGCAGCAGACGCTGCACCTGCTCAG
GCAGAAGAGAAGAAAGAAGCAGCTCCGGCAGCAGCACCAGCG
GCTGCGGCGGCAAAAGACGTTAACGTTCCGGATATCGGCAGCG
ACGAAGTTGAAGTGACCGAAATCCTGGTGAAAGTTGGCGATAA
AGTTGAAGCTGAACAGTCGCTGATCACCGTAGAAGGCGACAAG
GCTTCTATGGAAGTTCCGGCTCCGTTTGCTGGCACCGTGAAAGA
GATCAAAGTGAACGTGGGTGACAAAGTGTCTACCGGCTCGCTG
ATTATGGTCTTCGAAGTCGCGGGTGAAGCAGGCGCGGCAGCTCC
GGCCGCTAAACAGGAAGCAGCTCCGGCAGCGGCCCCTGCACCA
GCGGCTGGCGTGAAAGAAGTTAACGTTCCGGATATCGGCGGTG
ACGAAGTTGAAGTGACTGAAGTGATGGTGAAAGTGGGCGACAA
AGTTGCCGCTGAACAGTCACTGATCACCGTAGAAGGCGACAAA
GCTTCTATGGAAGTTCCGGCGCCGTTTGCAGGCGTCGTGAAGGA
aceF
ACTGAAAGTCAACGTTGGCGATAAAGTGAAAACTGGCTCGCTG
SEQ ID NO: 33 ATTATGATCTTCGAAGTTGAAGGCGCAGCGCCTGCGGCAGCTCC
TGCGAAACAGGAAGCGGCAGCGCCGGCACCGGCAGCAAAAGCT
GAAGCCCCGGCAGCAGCACCAGCTGCGAAAGCGGAAGGCAAAT
CTGAATTTGCTGAAAACGACGCTTATGTTCACGCGACTCCGCTG
ATCCGCCGTCTGGCACGCGAGTTTGGTGTTAACCTTGCGAAAGT
GAAGGGCACTGGCCGTAAAGGTCGTATCCTGCGCGAAGACGTT
CAGGCTTACGTGAAAGAAGCTATCAAACGTGCAGAAGCAGCTC
CGGCAGCGACTGGCGGTGGTATCCCTGGCATGCTGCCGTGGCCG
AAGGTGGACTTCAGCAAGTTTGGTGAAATCGAAGAAGTGGAAC
TGGGCCGCATCCAGAAAATCTCTGGTGCGAACCTGAGCCGTAAC
TGGGTAATGATCCCGCATGTTACTCACTTCGACAAAACCGATAT
CACCGAGTTGGAAGCGTTCCGTAAACAGCAGAACGAAGAAGCG
GCGAAACGTAAGCTGGATGTGAAGATCACCCCGGTTGTCTTCAT
CATGAAAGCCGTTGCTGCAGCTCTTGAGCAGATGCCTCGCTTCA
ATAGTTCGCTGTCGGAAGACGGTCAGCGTCTGACCCTGAAGAAA
TACATCAACATCGGTGTGGCGGTGGATACCCCGAACGGTCTGGT
TGTTCCGGTATTCAAAGACGTCAACAAGAAAGGCATCATCGAGC
TGTCTCGCGAGCTGATGACTATTTCTAAGAAAGCGCGTGACGGT

Gene sequence Description AAGCTGACTGCGGGCGAAATGCAGGGCGGTTGCTTCACCATCTC
CAGCATCGGCGGCCTGGGTACTACCCACTTCGCGCCGATTGTGA
ACGCGCCGGAAGTGGCTATCCTCGGCGTTTCCAAGTCCGCGATG
GAGCCGGTGTGGAATGGTAAAGAGTTCGTGCCGCGTCTGATGCT
GCCGATTTCTCTCTCCTTCGACCACCGCGTGATCGACGGTGCTG
ATGGTGCCCGTTTCATTACCATCATTAACAACACGCTGTCTGAC
ATTCGCCGTCTGGTGATGTAA
ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAG
GCCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGATTTAGGTC
TGGAAACCGTAATCGTAGAACGTTACAACACCCTTGGCGGTGTT
TGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGT
AGCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGT
ATCGTCTTCGGCGAACCGAAAACCGATATCGACAAGATTCGTAC
CTGGAAAGAGAAAGTGATCAATCAGCTGACCGGTGGTCTGGCT
GGTATGGCGAAAGGCCGCAAAGTCAAAGTGGTCAACGGTCTGG
GTAAATTCACCGGGGCTAACACCCTGGAAGTTGAAGGTGAGAA
CGGCAAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCG
GGTTCTCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCC
GCGTATCTGGGACTCCACTGACGCGCTGGAACTGAAAGAAGTA
CCAGAACGCCTGCTGGTAATGGGTGGCGGTATCATCGGTCTGGA
AATGGGCACCGTTTACCACGCGCTGGGTTCACAGATTGACGTGG
TTGAAATGTTCGACCAGGTTATCCCGGCAGCTGACAAAGACATC
GTTAAAGTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGAT
1pd GCTGGAAACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGC
SEQ ID NO: 34 ATTTATGTGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGC
AGCGTTACGACGCCGTGCTGGTAGCGATTGGTCGTGTGCCGAAC
GGTAAAAACCTCGACGCAGGCAAAGCAGGCGTGGAAGTTGACG
ACCGTGGTTTCATCCGCGTTGACAAACAGCTGCGTACCAACGTA
CCGCACATCTTTGCTATCGGCGATATCGTCGGTCAACCGATGCT
GGCACACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTA
TCGCCGGTAAGAAACACTACTTCGATCCGAAAGTTATCCCGTCC
ATCGCCTATACCAAACCAGAAGTTGCATGGGTGGGTCTGACTGA
GAAAGAAGCGAAAGAGAAAGGCATCAGCTATGAAACCGCCACC
TTCCCGTGGGCTGCTTCTGGTCGTGCTATCGCTTCCGACTGCGCA
GACGGTATGACCAAGCTGATTTTCGACAAAGAATCTCACCGTGT
GATCGGTGGTGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGG
GTGAAATCGGCCTGGCAATCGAAATGGGTTGTGATGCTGAAGA
CATCGCACTGACCATCCACGCGCACCCGACTCTGCACGAGTCTG
TGGGCCTGGCGGCAGAAGTGTTCGAAGGTAGCATTACCGACCTG
CCGAACCCGAAAGCGAAGAAGAAGTAA
ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA
AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTA
tesB GGTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTT
SEQ ID NO: 10 GTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATT
CGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCG
ATTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAG

Gene sequence Description CGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTT
ATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACAT
CAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTC
GGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCA
GTGCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCG
TCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAAC
CACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGAT
GACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTT
AACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCT
CGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGT
TCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTG
GAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGA
GTTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGG
AAGGGGTGATGCGTAATCACAATTAA
ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGT
CTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGAC
GTTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGC
CCTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGAT
GGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCC
ACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGG
GGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTC
GCGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGAC
TTACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGA
TGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGT
AATGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCT
acul CCGTTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCG
SEQ =ID NO: 35 GCAGTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTT
GGGGGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAG
GTACGCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGT
GGCGGGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGT
ATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGAT
CTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTG
GCAGGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGC
AGCGTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGG
AGGAGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGAC
AGCACCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTA
TCCCCGTAACGCCCTAA
Table 5. Propionate Cassette Sequences Sleeping Beauty Operon Sbm ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAA
SEQ ID NO: 36 TTGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGTTCATCAAA
CCGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCG
ATCTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCC
GCCCTACGTTCGTGGCCCGCGTGCCACTATGTATACCGCCCAA
CCGTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAG

AGTCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAA
AGGTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTAC
GACTCCGATAACCCGCGCGTGGCGGGCGACGTCGGCAAAGCG
GGCGTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCG
ACCAGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAA
TGGCGCAGTGCTACCAGTACTGGCGTTTTATATCGTCGCCGCA
GAAGAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATT
CAAAACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATA
TTTACCCACCAAAACCGTCAATGCGCATTATCGCCGACATCAT
CGCCTGGTGTTCCGGCAACATGCCGCGATTTAATACCATCAGT
ATCAGCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTG
CAGCAGGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCA
AAGCAGCAATCTCTGCCGGACTGAAAATTGATGACTTCGCTCC
TCGCCTGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGA
ACGTCGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGC
GGTCAGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCT
GCGTACCCACTGCCAGACCTCAGGCTGGAGCCTGACTGAACA
GGATCCGTATAACAACGTTATCCGCACCACCATTGAAGCGCTG
GCTGCGACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCT
TTGACGAAGCGCTTGGTTTGCCTACCGATTTCTCAGCACGCAT
TGCCCGCAACACCCAGATCATCATCCAGGAAGAATCAGAACT
CTGCCGCACCGTCGATCCACTGGCCGGATCCTATTACATTGAG
TCGCTGACCGATCAAATCGTCAAACAAGCCAGAGCTATTATCC
AACAGATCGACGAAGCCGGTGGCATGGCGAAAGCGATCGAAG
CAGGTCTGCCAAAACGAATGATCGAAGAGGCCTCAGCGCGCG
AACAGTCGCTGATCGACCAGGGCAAGCGTGTCATCGTTGGTGT
CAACAAGTACAAACTGGATCACGAAGACGAAACCGATGTACT
TGAGATCGACAACGTGATGGTGCGTAACGAGCAAATTGCTTC
GCTGGAACGCATTCGCGCCACCCGTGATGATGCCGCCGTAACC
GCCGCGTTGAACGCCCTGACTCACGCCGCACAGCATAACGAA
AACCTGCTGGCTGCCGCTGTTAATGCCGCTCGCGTTCGCGCCA
CCCTGGGTGAAATTTCCGATGCGCTGGAAGTCGCTTTCGACCG
TTATCTGGTGCCAAGCCAGTGTGTTACCGGCGTGATTGCGCAA
AGCTATCATCAGTCTGAGAAATCGGCCTCCGAGTTCGATGCCA
TTGTTGCGCAAACGGAGCAGTTCCTTGCCGACAATGGTCGTCG
CCCGCGCATTCTGATCGCTAAGATGGGCCAGGATGGACACGA
TCGCGGCGCGAAAGTGATCGCCAGCGCCTATTCCGATCTCGGT
TTCGACGTAGATTTAAGCCCGATGTTCTCTACACCTGAAGAGA
TCGCCCGCCTGGCCGTAGAAAACGACGTTCACGTAGTGGGCG
CATCCTCACTGGCTGCCGGTCATAAAACGCTGATCCCGGAACT
GGTCGAAGCGCTGAAAAAATGGGGACGCGAAGATATCTGCGT
GGTCGCGGGTGGCGTCATTCCGCCGCAGGATTACGCCTTCCTG
CAAGAGCGCGGCGTGGCGGCGATTTATGGTCCAGGTACACCT
ATGCTCGACAGTGTGCGCGACGTACTGAATCTGATAAGCCAGC
ATCATGATTAA
ygfD ATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCTTAC
SEQ ID NO: 37 GTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGG
TGGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGC

TGCTTGATGCCATTATGCCGTACTGCGGTAACACCCTGCGACT
GGGCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTT
GAGGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCG
CGGTTATTGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAG
CATTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTGCC
GAAGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGG
GCGGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCG
AAGCAGCGGGTTATGACGTAGTGATTGTCGAAACGGTTGGCG
TCGGGCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTT
TTATCTCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGG
CATTAAAAAAGGGCTGATGGAAGTGGCTGATCTGATCGTTATC
AACAAAGACGATGGCGATAACCATACCAATGTCGCCATTGCC
CGGCATATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAAT
ACGACGAATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGG
AAAAACGTGGAATCGATGAGATCTGGCACGCCATCATCGACT
TCAAAACCGCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCG
GCAACAACAATCGGTGGAATGGCTGCGTAAGCAGACCGAAGA
AGAAGTACTGAATCACCTGTTCGCGAATGAAGATTTCGATCGC
TATTACCGCCAGACGCTTTTAGCGGTCAAAAACAATACGCTCT
CACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCCAGAC
GCAATATTTTGATTAA
ygfG ATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGG
SEQ ID NO: 38 CGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAG
TAAAGTCTTTATTGATGATCTTATGCAGGCGTTAAGCGATCTC
AACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTG
GATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCC
GTCTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGT
CAAATCACCCGCATGATCCAAAAATTCCCGAAACCGATCATTT
CGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGAT
CATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTC
TCAATGACGCCTGTAAACCTCGGCGTCCCGTATAACCTGGTCG
GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA
AGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCG
CTGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAAC
TGGAAGATTTCACCTTACAAATGGCGCACCACATCTCTGAGAA
AGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTA
CTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGT
ATTCAGGGGATGCGCCGCGCGGTGTATGACAGCGAAGATTAC
CAGGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAAT
TTCGTTGGTCATTAA
ygfH ATGGAAACTCAGTGGACAAGGATGACCGCCAATGAAGCGGCA
SEQ ID NO: 39 GAAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTA
CCCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCG
CAGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAAT
TCGCCTTCTGACGGGTGCGTCAATCAGCGCCGCCGCTGACGAT
GTACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATC
AAACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGG
TGAGTTTCGTTGACCTGCATTTGAGCGAAGTGGCGCAAATGGT

CAATTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAA
GCATCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGC
GGGATCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAA
GTGATCATTGAACTCAATCACTATCACGATCCGCGCGTTGCAG
AACTGGCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAA
TAGCGTGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGC
TATGTGCAAATCGATCCGAAAAAGATTGTCGCCGTCGTGGAA
ACCAACTTGCCCGACGCCGGTAATATGCTGGATAAGCAAAAT
CCCATGTGCCAGCAGATTGCCGATAACGTGGTCACGTTCTTAT
TGCAGGAAATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCC
GCTGCAAAGTGGCGTGGGCAATATCAATAATGCGGTAATGGC
GCGTCTGGGGGAAAACCCGGTAATTCCTCCGTTTATGATGTAT
TCGGAAGTGCTACAGGAATCGGTGGTGCATTTACTGGAAACC
GGCAAAATCAGCGGGGCCAGCGCCTCCAGCCTGACAATCTCG
GCCGATTCCCTGCGCAAGATTTACGACAATATGGATTACTTTG
CCAGCCGCATTGTGTTGCGTCCGCAGGAGATTTCCAATAACCC
GGAAATCATCCGTCGTCTGGGCGTCATCGCTCTGAACGTCGGC
CTGGAGTTTGATATTTACGGGCATGCCAACTCAACACACGTAG
CCGGGGTCGATCTGATGAACGGCATCGGCGGCAGCGGTGATT
TTGAACGCAACGCGTATCTGTCGATCTTTATGGCCCCGTCGAT
TGCTAAAGAAGGCAAGATCTCAACCGTCGTGCCAATGTGCAG
CCATGTTGATCACAGCGAACACAGCGTCAAAGTGATCATCACC
GAACAAGGGATCGCCGATCTGCGCGGTCTTTCCCCGCTTCAAC
GCGCCCGCACTATCATTGATAATTGTGCACATCCTATGTATCG
GGATTATCTGCATCGCTATCTGGAAAATGCGCCTGGCGGACAT
ATTCACCACGATCTTAGCCACGTCTTCGACTTACACCGTAATTT
AATTGCAACCGGCTCGATGCTGGGTTAA
Table 6. Sequences of Propionate Cassette from Propioni Bacteria Description Sequence mutA ATGAGCAGCACGGATCAGGGGACCAACCCCGCCGACACTGAC
SEQ ID NO: 40 GACCTCACTCCCACCACACTCAGTCTGGCCGGGGATTTCCCCA
AGGCCACTGAGGAGCAGTGGGAGCGCGAAGTTGAGAAGGTAT
TCAACCGTGGTCGTCCACCGGAGAAGCAGCTGACCTTCGCCGA
GTGTCTGAAGCGCCTGACGGTTCACACCGTCGATGGCATCGAC
ATCGTGCCGATGTACCGTCCGAAGGACGCGCCGAAGAAGCTG
GGTTACCCCGGCGTCACCCCCTTCACCCGCGGCACCACGGTGC
GCAACGGTGACATGGATGCCTGGGACGTGCGCGCCCTGCACG
AGGATCCCGACGAGAAGTTCACCCGCAAGGCGATCCTTGAAG
ACCTGGAGCGTGGCGTCACCTCCCTGTTGTTGCGCGTTGATCC
CGACGCGATCGCACCCGAGCACCTCGACGAGGTCCTCTCCGAC
GTCCTGCTGGAAATGACCAAGGTGGAGGTCTTCAGCCGCTACG
ACCAGGGTGCCGCCGCCGAGGCCTTGATGGGCGTCTACGAGC
GCTCCGACAAGCCGGCGAAGGACCTGGCCCTGAACCTGGGCC
TGGATCCCATCGGCTTCGCGGCCCTGCAGGGCACCGAGCCGG
ATCTGACCGTGCTCGGTGACTGGGTGCGCCGCCTGGCGAAGTT
CTCACCGGACTCGCGCGCCGTCACGATCGACGCGAACGTCTAC

CACAACGCCGGTGCCGGCGACGTGGCAGAGCTCGCTTGGGCA
CTGGCCACCGGCGCGGAGTACGTGCGCGCCCTGGTCGAACAG
GGCTTCAACGCCACAGAGGCCTTCGACACGATCAACTTCCGTG
TCACCGCCACCCACGACCAGTTCCTCACGATCGCCCGTCTTCG
CGCCCTGCGCGAGGCATGGGCCCGCATCGGCGAGGTCTTTGGC
GTGGACGAGGACAAGCGCGGCGCTCGCCAGAATGCGATCACC
AGTTGGCGTGAGCTCACCCGCGAAGACCCCTATGTCAACATCC
TTCGCGGTTCGATTGCCACCTTCTCCGCCTCCGTTGGCGGGGC
CGAGTCGATCACGACGCTGCCCTTCACCCAGGCCCTCGGCCTG
CCGGAGGACGACTTCCCGCTGCGCATCGCGCGCAACACGGGC
ATCGTGCTCGCCGAAGAGGTGAACATCGGCCGCGTCAACGAC
CCGGCCGGTGGCTCCTACTACGTCGAGTCGCTCACTCGCACCC
TGGCCGACGCTGCCTGGAAGGAATTCCAGGAGGTCGAGAAGC
TCGGTGGCATGTCGAAGGCGGTCATGACCGAGCACGTCACCA
AGGTGCTCGACGCCTGCAATGCCGAGCGCGCCAAGCGCCTGG
CCAACCGCAAGCAGCCGATCACCGCGGTCAGCGAGTTCCCGA
TGATCGGGGCCCGCAGCATCGAGACCAAGCCGTTCCCAACCG
CTCCGGCGCGCAAGGGCCTGGCCTGGCATCGCGATTCCGAGGT
GTTCGAGCAGCTGATGGATCGCTCCACCAGCGTCTCCGAGCGC
CCCAAGGTGTTCCTTGCCTGCCTGGGCACCCGTCGCGACTTCG
GTGGCCGCGAGGGCTTCTCCAGCCCGGTATGGCACATCGCCGG
TATCGACACCCCGCAGGTCGAAGGCGGCACCACCGCCGAGAT
CGTCGAGGCGTTCAAGAAGTCGGGCGCCCAGGTGGCCGATCT
CTGCTCGTCCGCCAAGATCTACGCGCAGCAGGGACTTGAGGTT
GCCAAGGCGCTCAAGGCCGCCGGCGCGAAGGCCCTGTATCTG
TCGGGCGCCTTCAAGGAGTTCGGCGATGACGCCGCCGAGGCC
GAGAAGCTGATCGACGGACGCCTGTACATGGGCATGGATGTC
GTCGACACCCTGTCCTCCACCCTTGATATCTTGGGAGTCGCGA
AGTGA
mutB GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTGGGCAATG
SEQ ID NO: 41 CCCCGGTTCCTGCTGATGCCGCACAGCGCTTCGAGGAGTTGGC
CGCCAAGGCCGGCACCGAAGAGGCGTGGGAGACGGCTGAGCA
GATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGAC
ATGGACTGGCTGGACACCTACGCCGGTATCCCGCCGTTCGTCC
ACGGCCCATATGCAACCATGTACGCGTTCCGTCCCTGGACGAT
TCGCCAGTACGCCGGCTTCTCCACGGCCAAGGAGTCCAACGCC
TTCTACCGCCGCAACCTTGCGGCGGGCCAGAAGGGCCTGTCGG
TTGCCTTCGACCTGCCCACCCACCGCGGCTACGACTCGGACAA
TCCCCGCGTCGCCGGTGACGTCGGCATGGCCGGGGTGGCCATC
GACTCCATCTATGACATGCGCGAGCTGTTCGCCGGCATTCCGC
TGGACCAGATGAGCGTGTCGATGACCATGAACGGCGCCGTGC
TGCCGATCCTGGCCCTCTATGTGGTGACCGCCGAGGAGCAGGG
CGTCAAGCCCGAGCAGCTCGCCGGGACGATCCAGAACGACAT
CCTCAAGGAGTTCATGGTTCGTAACACCTATATCTACCCGCCG
CAGCCGAGTATGCGAATCATCTCCGAGATCTTCGCCTACACGA
GTGCCAATATGCCGAAGTGGAATTCGATTTCCATTTCCGGCTA
CCACATGCAGGAAGCCGGCGCCACGGCCGACATCGAGATGGC
CTACACCCTGGCCGACGGTGTCGACTACATCCGCGCCGGCGAG

TCGGTGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCTGTCCT
TCTTCTGGGGCATCGGCATGAACTTCTTCATGGAGGTTGCCAA
GCTGCGTGCCGCACGTATGTTGTGGGCCAAGCTGGTGCATCAG
TTCGGGCCGAAGAATCCGAAGTCGATGAGCCTGCGCACCCAC
TCGCAGACCTCCGGTTGGTCGCTGACCGCCCAGGACGTCTACA
ACAACGTCGTGCGTACCTGCATCGAGGCCATGGCCGCCACCCA
GGGCCATACCCAGTCGCTGCACACGAACTCGCTCGACGAGGC
CATTGCCCTACCGACCGATTTCAGCGCCCGCATCGCCCGTAAC
ACCCAGCTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTG
ATCGACCCGTGGAGCGGCTCGGCATACGTCGAGGAGCTCACC
TGGGACCTGGCCCGCAAGGCATGGGGCCACATCCAGGAGGTC
GAGAAGGTCGGCGGCATGGCCAAGGCCATCGAAAAGGGCATC
CCCAAGATGCGCATTGAGGAAGCCGCCGCCCGCACCCAGGCA
CGCATCGACTCCGGCCGTCAGCCGCTGATCGGCGTGAACAAGT
ACCGCCTGGAGCACGAGCCGCCGCTCGATGTGCTCAAGGTTG
ACAACTCCACGGTGCTCGCCGAGCAGAAGGCCAAGCTGGTCA
AGCTGCGCGCCGAGCGCGATCCCGAGAAGGTCAAGGCCGCCC
TCGACAAGATCACCTGGGCTGCCGCCAACCCCGACGACAAGG
ATCCGGATCGCAACCTGCTGAAGCTGTGCATCGACGCTGGCCG
CGCCATGGCGACGGTCGGCGAGATGAGCGACGCGCTCGAGAA
GGTCTTCGGACGCTACACCGCCCAGATTCGCACCATCTCCGGT
GTGTACTCGAAGGAAGTGAAGAACACGCCTGAGGTTGAGGAA
GCACGCGAGCTCGTTGAGGAATTCGAGCAGGCCGAGGGCCGT
CGTCCTCGCATCCTGCTGGCCAAGATGGGCCAGGACGGTCACG
ACCGTGGCCAGAAGGTCATCGCCACCGCCTATGCCGACCTCGG
TTTCGACGTCGACGTGGGCCCGCTGTTCCAGACCCCGGAGGAG
ACCGCACGTCAGGCCGTCGAGGCCGATGTGCACGTGGTGGGC
GTTTCGTCGCTCGCCGGCGGGCATCTGACGCTGGTTCCGGCCC
TGCGCAAGGAGCTGGACAAGCTCGGACGTCCCGACATCCTCA
TCACCGTGGGCGGCGTGATCCCTGAGCAGGACTTCGACGAGCT
GCGTAAGGACGGCGCCGTGGAGATCTACACCCCCGGCACCGT
CATTCCGGAGTCGGCGATCTCGCTGGTCAAGAAACTGCGGGCT
TCGCTCGATGCCTAG
GI:18042134 ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCAT
ATGCGTGCCCCGACGCCGACGAGGCTTCCAAGTACTACCAGG
SEQ ID NO: 42 AGACCTTCGGCTGGCATGAGCTCCACCGCGAGGAGAACCCGG
AGCAGGGAGTCGTCGAGATCATGATGGCCCCGGCTGCGAAGC
TGACCGAGCACATGACCCAGGTTCAGGTCATGGCCCCGCTCAA
CGACGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACAATGG
TCGCGCCGGACTGCACCACATGGCATGGCGTGTCGATGACATC
GACGCCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTG
CTGTATGACGAGCCCAAGCTCGGCACCGGCGGCAACCGCATC
AACTTCATGCATCCCAAGTCGGGCAAGGGCGTGCTCATCGAGC
TCACCCAGTACCCGAAGAACTGA
mmdA ATGGCTGAAAACAACAATTTGAAGCTCGCCAGCACCATGGAA
GGTCGCGTGGAGCAGCTCGCAGAGCAGCGCCAGGTGATCGAA
SEQ ID NO: 43 GCCGGTGGCGGCGAACGTCGCGTCGAGAAGCAACATTCCCAG

GGTAAGCAGACCGCTCGTGAGCGCCTGAACAACCTGCTCGAT
CCCCATTCGTTCGACGAGGTCGGCGCTTTCCGCAAGCACCGCA
CCACGTTGTTCGGCATGGACAAGGCCGTCGTCCCGGCAGATGG
CGTGGTCACCGGCCGTGGCACCATCCTTGGTCGTCCCGTGCAC
GCCGCGTCCCAGGACTTCACGGTCATGGGTGGTTCGGCTGGCG
AGACGCAGTCCACGAAGGTCGTCGAGACGATGGAACAGGCGC
TGCTCACCGGCACGCCCTTCCTGTTCTTCTACGATTCGGGCGG
CGCCCGGATCCAGGAGGGCATCGACTCGCTGAGCGGTTACGG
CAAGATGTTCTTCGCCAACGTGAAGCTGTCGGGCGTCGTGCCG
CAGATCGCCATCATTGCCGGCCCCTGTGCCGGTGGCGCCTCGT
ATTCGCCGGCACTGACTGACTTCATCATCATGACCAAGAAGGC
CCATATGTTCATCACGGGCCCCCAGGTCATCAAGTCGGTCACC
GGCGAGGATGTCACCGCTGACGAACTCGGTGGCGCTGAGGCC
CATATGGCCATCTCGGGCAATATCCACTTCGTGGCCGAGGACG
ACGACGCCGCGGAGCTCATTGCCAAGAAGCTGCTGAGCTTCCT
TCCGCAGAACAACACTGAGGAAGCATCCTTCGTCAACCCGAA
CAATGACGTCAGCCCCAATACCGAGCTGCGCGACATCGTTCCG
ATTGACGGCAAGAAGGGCTATGACGTGCGCGATGTCATTGCC
AAGATCGTCGACTGGGGTGACTACCTCGAGGTCAAGGCCGGC
TATGCCACCAACCTCGTGACCGCCTTCGCCCGGGTCAATGGTC
GTTCGGTGGGCATCGTGGCCAATCAGCCGTCGGTGATGTCGGG
TTGCCTCGACATCAACGCCTCTGACAAGGCCGCCGAATTCGTG
AATTTCTGCGATTCGTTCAACATCCCGCTGGTGCAGCTGGTCG
ACGTGCCGGGCTTCCTGCCCGGCGTGCAGCAGGAGTACGGCG
GCATCATTCGCCATGGCGCGAAGATGCTGTACGCCTACTCCGA
GGCCACCGTGCCGAAGATCACCGTGGTGCTCCGCAAGGCCTA
CGGCGGCTCCTACCTGGCCATGTGCAACCGTGACCTTGGTGCC
GACGCCGTGTACGCCTGGCCCAGCGCCGAGATTGCGGTGATG
GGCGCCGAGGGTGCGGCAAATGTGATCTTCCGCAAGGAGATC
AAGGCTGCCGACGATCCCGACGCCATGCGCGCCGAGAAGATC
GAGGAGTACCAGAACGCGTTCAACACGCCGTACGTGGCCGCC
GCCCGCGGTCAGGTCGACGACGTGATTGACCCGGCTGATACCC
GTCGAAAGATTGCTTCCGCCCTGGAGATGTACGCCACCAAGCG
TCAGACCCGCCCGGCGAAGAAGCATGGAAACTTCCCCTGCTG
A

SEQ ID NO: 44 GGTATCACCGAGCTCGTGCTGCGCGATGCCCATCAGAGCCTGA
TGGCCACACGAATGGCAATGGAAGACATGGTCGGCGCCTGTG
CAGACATTGATGCTGCCGGGTACTGGTCAGTGGAGTGTTGGGG
TGGTGCCACGTATGACTCGTGTATCCGCTTCCTCAACGAGGAT
CCTTGGGAGCGTCTGCGCACGTTCCGCAAGCTGATGCCCAACA
GCCGTCTCCAGATGCTGCTGCGTGGCCAGAACCTGCTGGGTTA
CCGCCACTACAACGACGAGGTCGTCGATCGCTTCGTCGACAAG
TCCGCTGAGAACGGCATGGACGTGTTCCGTGTCTTCGACGCCA
TGAATGATCCCCGCAACATGGCGCACGCCATGGCTGCCGTCAA
GAAGGCCGGCAAGCACGCGCAGGGCACCATTTGCTACACGAT
CAGCCCGGTCCACACCGTTGAGGGCTATGTCAAGCTTGCTGGT
CAGCTGCTCGACATGGGTGCTGATTCCATCGCCCTGAAGGACA

TGGCCGCCCTGCTCAAGCCGCAGCCGGCCTACGACATCATCAA
GGCCATCAAGGACACCTACGGCCAGAAGACGCAGATCAACCT
GCACTGCCACTCCACCACGGGTGTCACCGAGGTCTCCCTCATG
AAGGCCATCGAGGCCGGCGTCGACGTCGTCGACACCGCCATC
TCGTCCATGTCGCTCGGCCCGGGCCACAACCCCACCGAGTCGG
TTGCCGAGATGCTCGAGGGCACCGGGTACACCACCAACCTTG
ACTACGATCGCCTGCACAAGATCCGCGATCACTTCAAGGCCAT
CCGCCCGAAGTACAAGAAGTTCGAGTCGAAGACGCTTGTCGA
CACCTCGATCTTCAAGTCGCAGATCCCCGGCGGCATGCTCTCC
AACATGGAGTCGCAGCTGCGCGCCCAGGGCGCCGAGGACAAG
ATGGACGAGGTCATGGCAGAGGTGCCGCGCGTCCGCAAGGCC
GCCGGCTTCCCGCCCCTGGTCACCCCGTCCAGCCAGATCGTCG
GCACGCAGGCCGTGTTCAACGTGATGATGGGCGAGTACAAGA
GGATGACCGGCGAGTTCGCCGACATCATGCTCGGCTACTACGG
CGCCAGCCCGGCCGATCGCGATCCGAAGGTGGTCAAGTTGGC
CGAGGAGCAGTCCGGCAAGAAGCCGATCACCCAGCGCCCGGC
CGATCTGCTGCCCCCCGAGTGGGAGGAGCAGTCCAAGGAGGC
CGCGGCCCTCAAGGGCTTCAACGGCACCGACGAGGACGTGCT
CACCTATGCACTGTTCCCGCAGGTCGCTCCGGTCTTCTTCGAG
CATCGCGCCGAGGGCCCGCACAGCGTGGCTCTCACCGATGCCC
AGCTGAAGGCCGAGGCCGAGGGCGACGAGAAGTCGCTCGCCG
TGGCCGGTCCCGTCACCTACAACGTGAACGTGGGCGGAACCG
TCCGCGAAGTCACCGTTCAGCAGGCGTGA
Bccp ATGAAACTGAAGGTAACAGTCAACGGCACTGCGTATGACGTT
GACGTTGACGTCGACAAGTCACACGAAAACCCGATGGGCACC
SEQ ID NO: 45 ATCCTGTTCGGCGGCGGCACCGGCGGCGCGCCGGCACCGCGC
GCAGCAGGTGGCGCAGGCGCCGGTAAGGCCGGAGAGGGCGA
GATTCCCGCTCCGCTGGCCGGCACCGTCTCCAAGATCCTCGTG
AAGGAGGGTGACACGGTCAAGGCTGGTCAGACCGTGCTCGTT
CTCGAGGCCATGAAGATGGAGACCGAGATCAACGCTCCCACC
GACGGCAAGGTCGAGAAGGTCCTTGTCAAGGAGCGTGACGCC
GTGCAGGGCGGTCAGGGTCTCATCAAGATCGGCTGA
[0280] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ
ID NO: 10) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ ID NO: 10) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ ID

NO: 10) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21- SEQ ID NO: 35, and SEQ ID NO: 10) or a functional fragment thereof.
[0281] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 5 (SEQ ID NO: 36- SEQ ID NO: 39) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 5 (SEQ
ID NO:
36- SEQ ID NO: 39) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%
homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 5 (SEQ ID
NO: 36-SEQ ID NO: 39) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 5 (SEQ ID NO: 36- SEQ ID NO: 39) or a functional fragment thereof.
[0282] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 6 (SEQ ID NO: 40- SEQ ID NO: 45) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 6 (SEQ
ID NO:
40- SEQ ID NO: 45) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%
homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 6 (SEQ ID
NO: 40-SEQ ID NO: 45) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 6 (SEQ ID NO: 40- SEQ ID NO: 45) or a functional fragment thereof.
[0283] Table 7 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria.

Table 7. Polypeptide Sequences for Propionate Synthesis Pct MRKVPIITADEAAKLIKDGDTvriTSGINGN AIPEALDR AVEKRFLETGE
SEQ ID PKNITYVYCGS QGNRDGRGAEHFAHEGLLKRYIAGHWATVPALG KM
NO: 46 AMENKMEAYN VSQG ALCHLERDIASHKPGVFTKV GIGTHDPRNGGG
KVNDITKEDIVEINEIKGQEYLFYPAFPIEVAIJRGTYADESGNITFEKE
VAPLEGTSVCQAVKNSGGIVVVQVERVVKAGTLDPRFIVKVPGIYVDY
VVADPEDHQQSLDCEYDPALSGEHRRPEVNIGEPLPLS NKKVIGRRG A
IELEKDVAVNLGVGAPEYVASVADEEGTVDFMTLTAESGAIGGVPAGG
VRFGASYNADALIDQGY-QFDY-YDGGGLDLCYLGLAECDEKGNINVSR
FGPRIAGCGGFINITQNTPKVEFCGTFT AGGLKVKIEDG KVIIVQEGKQK
KFLKAVEQITFNGDVALANKQQVTYITERCVFLLKEDGLHLSEIAPGID
LQTQILDVMDFAPIIDRDANGQIKLMDAALFAEGLMGLKEMKS*
lcdA MSLTQGMKAKQLLAYFQG KADQD AREAKARGELVCWS AS V APPEFC
SEQ ID VI MGIAMIYPEffiAAGIG ARKGAMD NILE V ADRKGYN VDCCS YGRVN-NO: 47 MGYMECLKEAAITGVKPEVINNSPAADVPLPDLVITCNNICNTLLKWY
ENLAAELDIPCIVIDVPFNITIMPIPEYAKAYIADQFRNAISQLEVICGRPF
DWKKFKEV KDQTQRS V YIIWNRIAENTAKYKPSPLNGFDLFNYMALIV
ACRSLDYAEITFKAFADELEENLKAGIYAFKGAEKTRFQWEGIAVWPH
LGHTFKSMKNILNSIMTGTAYPALWDLHYDANDESMHS MAE AYTRIYI
NTCLQNKVEVIJLGIMEKGQVDGTVYIILNRSCKLMS FLNVETAEIIKEK
NGLPYVSIDGDQTDPRVFSPAQFDTRVQALVEMMEANMAAAE*
lcdB MS RVEAILS QLKDVAANPKKAMDDY KAETGKGAVGIMPIYS PEEMVH
SEQ ID AAGYLPMGIWG AQGKTISKARTYLPAFACSVMQQVMELQCEGAYDD
NO: 48 LS AVMS VPCDTLI(CLSQKWKGTSPVIVFTI-IPQNRGLE AANQFLVTEYE
LV :KAQLES VLGV KIS NAALENSIAIYNENRAVMREFV KV AADYPQVID
AVSRHAVF KARQFMLKE KHT Al VKELIAE IKA1PVQPWDGKKVVVR3 ILLEPNELLDIFNEFKIAIVDDDLAQES RQIRVDVLDGEGGPLYRMAKA
-WQQMYGCS LA'FDTKKGRGRMLINKFIQTGADAIVV AMMKFCDPEEW-DYPVMYREFEEKGVKSLMIEVDQEVSSFEQIKTRLQSFVEML*
lcdC M Y IIGIDVGS ASS K AVII KDGKINV AAEVVQVG`FGSSGPQRALDKAFEV
SEQ ID S GLKKEDIS YTVATGYGRFNFS DAD KQIS EISCHAKGIYFLVPTARTIIDIG
NO: 49 GQDAKAIRLDD KGGIKQFFMND KC AAGTGRFLE V MARVLEFFLDEMAE
LDEQATDTAPISSTCTVFAESEVISQLSNGVSRNMIKGVHLSVASRACGL
AYRGGLEKDVVMTGGVAKNAGVVRAVAGVLKTDVIVAPNPQTTGALG
AMA AY EAAQ K KX
etfA MAFNS ADINS FRDIW VEVEQREGKLIN'fDFELISEGRKLADERGS KING
SEQ ID ILLGHEVEEIAKELGGYGADKVIVCDIVELKFYTTDAY AKVLCDVVME
NO: 50 EKPEVILIGATNIGRDLGPRCAARLHTGLTADCTHLDIDMN KYVDFLST
SSTLDISSMTFPMEDTNLKMTRPAFGGEILMATIICPRERPCMSTVRPGV
MKKAEFSQEMAQACQVVTRHVNLSDEDLKTKVINIVKETKKIVDLIGA
:EfIVS VGRGISKDVQGGIALAEKLADAFGNGVVGGSRAVIDSGWLPAD
FIQVGQTGKTVI-IPKVYNALGISGAIQI-IKAGMQDSELIIAVNKDETAPIF
DCADYGITGDLFKIVPMMIDAIKEGKNA*
acrB MRIYVCVKQVPDTSGKVAVNPDGTLNRASMAAHNPDDMSAIEQALKL
SEQ ID KDETGCQVTALTMGPPPAEGMLREHAMGADDGVLIS AREFGGSDTFA
NO: 51 TSQRSAAILIKLGLS NEDMIFCGRQAIDGDTAQVGPQIAEKLSIPQVTYG
AGIKKSGDINLVKRMLEDGYMMIEVETPCLITCIQDKAVKPRYMTLN

GIMECYS KPLLVLDYEALKDEPLIELDTIGLKGS PTNIFKS FTPPQKGVG
VMLQGTDKEKVEDLVDKLMQKHVI*
acrC MFLLKIKKERMKRMDFSLTREQEMLICKLARQFAEIELEPV AEEIDREH
SEQ ID VFRAENFKKNIAEIGLTGICTIPKEFGGSGGGTLEKYIAVSEFG KKCMAS A
NO: 52 S ILSIHLIAPQAIY KYGT KEQKETYLPRLTKGGELGAFALTEPNAGS DAG
AV KTTAILDSQTNEYVIAGT KCHISGGGRAGVINIFALTEPICKGLKGM
SAIIVEKGTPGFSIGKVESKMGIAGSETAELIFEDCRVPAANLLG KEGKG
KIAMEALDGARIGVG AQAIG IAEGAID LS V:KYVHERIQFGKPI AN LQGI
QWYIADM ATKTAA ARALVE FAAYLEDAGKPFTKIES AMCKLNAS EN A
RFVTNLALQIHGGYGYMKDYPLERMYRDAKITEIYEGTSEIHKVVIAR
:EVMKR*
thrAfbr MRVLKFGGTSVANAERFLRV AD1LESNARQGQVATVIS APAKITNHL V
SEQ ID AMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKTFVDQEF
NO: 53 AQIKHVLHGISLLGQCPDS INAALICRGEKMS IAIMAGVLEARGHNVTV
IDPVEKLLAVGHYLES TVDIAES TRRIA AS RIPADHMVLMAGFTAGNE K
GELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQVPD
ARLLKS MS YQEAMELS YFG AKVLHPRTITPIAQFQIPCLIKNTGNPQAP
GTLIGASRDEDELPV S NLNNM AMES VSGPGMKGM VG MAARVFA
AMSRARIS VLITQS SS EYSISFC VPQS DCVRAER AMQEEFYLEI ,KEGLL
:EPLAVIERLAIISVVGDGMRTLRGIS AKEFAALARANINIV AIAQRS S ER
SIS VVVNNDDATTGVRYTHQMLFNTDQVIEVINIGVGGVGGALLEQL
KRQQS WLKN KHIDLRVCGVANS KALLTNVHGLNLENWQEELAQAKE
PFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPN
KKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNA
GDELMKFSGILSGSLSYIFG KLDEGMS FSEATTLAREMGYTEPDPRDDL
SGMD ARKLLIL ARETGRE LELADIEIETVLPMEFN AEGDV AAFM ANLS
QLDDLFAARV AKARDEGKVLRY VGNIDEDGVCRVKIAEVDGNDPLFK
VKNGENALAFYSHYYQPLPLVLRGYGAGND VTAAGVFADLLRTLSW
KLGV*
thrB MVKVYAPASS ANMS VGFDVLGAAVTPVDGALLGDV VrtVEAAErfFSL
SEQ ID NNLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGS
NO: 54 GLGSSACSVVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYD
NVAPCF LGGMQLMIEENDIISQQVPGFDE WI WVL AYPGIKVSTAE AR' ILPAQYRRQDCIAHGRFILAGFIHACYS RQPELAAKLMKDVIAEPYRER
LLPGFRQARQ AVAEIGAVAS GIS GS GPTLFALCD KPETAQRVADWLGK
NYLQNQEGFVHICRLDTAGARVLEN*
thrC IVIKLYMKININEQVSFAQAVIQGLGKNQGLFFPLIDLPEFSUFEIDEML
SEQ ID KLDFVTRSAKILSAFIGDEINEILEERVRAAFAFPAPVANVESDVGCLE
NO: 55 LFHGPTLAFKDFGGRFMAQMLTHIAGDKPVT1LTATSGDTGAAVAHAF
YGLPNVKVVILYPRGKISPLQEKLFCTLGGNIET VAIDGDFDACQALVK
QAFDDEELKVALGLNS ANS INIS RLLAQICYYFEAVAQLPQETRNQLVV
S VPSGNEUDLTAGLLAKSLGLPV KRFIAATNVNDTVPRFLHDGQWSPK
ATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYAAVDDETTQ
QTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGTAHPAKFKE
S'VEAILGETLDLPKELAERADLPLLSEINLPADFAALR KLMNINHQ*
i/vAfbr MSETYVSEKSTGVMASGAELIRA ADIQTAQARISS VIAPTPLQYCPRLSE
SEQ ID ETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAAS AGNH
NO: 56 AQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVVIGNN

EDE AS AAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILS QLTS MG KS
ADHVMVPVGGGGLLAGVVS YMADMAPRTAIVGIEPAGAASMQAALH
NGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVIIMMSATEGAV
CTEMLDLYQNEGHAEPAGALSIAGLKEMSFAPGSAVVCIISGGNNDVL
RYAEIAIE:RSLVFIRGL KHYfINNFPQKPGQL RH FLEDILGPDDD1TLFEY
LKRNNRETGTALV GUMS E ASGLDS LLERMEESAIDS RRLEPGT PEYEY
LT*
ace MSERFPNDVDPIETRDWLQAIES VIREEGVERAQYLIDQLLAEARKGGV

NO: 57 KDLELGGHMASFQSS ATIYDVCFNHFFRARNEQDGGDLVYFQGHISPG
VYARAFLEGRLTQEQLDNFRQEVHGNGLSS YPHPKLMPEFWQFPTVS
MGLGPIGATYQ AKFLKYLEHRGL KDTS KQT V YAFLGDGEMDEPES KG
AITIATREKLDNLVEVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVI
KVM WGSRVYIDEIL RKDT SGKLIQLMNErtV:DGDYQTF KS KDGAYVREH
FFG KYPET AALVADWT DEQIWALNRGGHDPKKIY AAFKKAQETKGK
AT VfLAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVS
DADIEKLPYITFPEGSEEHTYLHAQRQ KLUGYLPSRQPNFTEKLELPS LQ
DFGALLEEQS KEISTTIAFVRALNVMLKN KS IKDRLVPHADE ARTFGME
GLFRQIGIYSPNGQQYTPQDREQVAYIKEDEKGQILQEGINELGAGCS
W LA AATS YS'f NNLPMIPFYIYYSMFGFQRIGDLCAVAAGDQQARGFLIG
GTSGRTTLNGEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGL
:ERMYGEKQENV YY \I TT LNENYHMPAMPEGAEEGIRKGIYKLETIEGS
KG KV QLLGSGS ILREVREAAEILAKDYGVGSDVYSVTSFTELARDGQD
CERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVP
ADDYRVLGTDGFGRSDS RE NLREIHFEVD AS YVVV AALGELAKRGEID
KKVVADAIAKFNIDADKVNPRI ,A*
aceF MAIETKVPDTGADEVEITEILV KV GD KVEAEQS UTVEGDKASME VPSPQ
SEQ ID AGIVKEIKVSVGDKTQFGALIMIEDSADGAADAAPAQAEEKKEAAPAA
NO: 58 APAAAAAKIWNVPDIGSDEVENTEILN KWH) KVEAEQS LITVEGDKAS
MEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAPAAKQE
AAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGD KY ANEQS LIT
VEGDKASMEVPAPFAGVVKELKVNVGD KY KTGRAMIFE VEGAAPAA
APAKQE AAAPAPAAKAEAPAAAPAAKAEG KS EFAENDAYVHATPLIR
RLARE FGVN LAKVKG`FGR KGRILREDV QAY VKE Al KR/ME AAPAATGG
GIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMIPHVTHFD
KTDITELEAFRKQQNEEAAKRKLDV KITPVVFIMKAVAAALEQMPRFN
SS LS EDGQRLTLICKYINIGVAVDTPNGLVVPVFKDVN KKGIIELSRELM
TISKKARDGKLTAGEMQGGCFTISSIGGLGTTHFAPIVNAPEVAILGVS K
SAMEPVWNGKEEVPRLMLPISLSFDHRVIDGADGARFITIINNTLSDIRR
LVM*
Lpd MSTE1 KTQVVVLGAGPAGYS AA FRCADLGL ETVIVERYNTLGGVUN
SEQ ID VGCIPS KALLHVAKVIEEAKALAEHGIVFGEPKTDID KIRTWKE KVINQ
NO: 59 LTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAII
NAGS RPIQLPFIPHEDPRIWDSTDALELKEVPERLLV MGGGIIGLEMGTV
YHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLETKVTAV
AKEDGIYVTMEGKKAPAEPQRYD AV LV Al GRVPNGKNLD AGKAGV-EVDDRGFIRVDKQLRTNVPHIFAIGDIVGQRMLAHKGVHEGHVAAEVI

SGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIENIG
CDAEDIALTIHAHPTLHESVGLAAEVFEGSTTDLPNPKAKKK*
tesB MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFGGQVVGQALYAA
SEQ ID KETVPEERLVHSFHS YFLRPGDSKKPHYDVETLRDGNSFSARRVAAIQ
NO: 20 NGKPIFYMTASFQAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLLP
PVL KD KfICDRPLEVRPVEFFINPLKGHVAEPHRQVWIR ANGSVPDDLR
VHQYLLGY ASDLNFLPVALQPHGIGFLEPGI QIATIDEISMWHIRPFNLN
EWLLYSVES'I'SASSARGFVRGEFY-TQDGVLVAS'f VQEGVMRNFIN*
acuI MRAVLIEKSDDTQS VS VTELAEDQLPEGDVINDVAYSTLNYKDALAIT
SEQ ID GKAPVV RRFPM \MGM FTGTVAQSS HADFKPGDRV1LNGWG VGE KIM
NO: 60 GGLAERARVRGDWLVPLPAPLDLRQAANIIGTAGYTAMLCVLALERH
GVVPGNGEIVVSGAAGGVGSVATTLLAAKGYEVAAVTGRASEAEYLR
GLGA AS-VIDRNEUFGKVRPLGQIER`vVAGGIDvAcisTryLANIALS
RGVVAACGLAAGMDLPASVAPFILRGMTLAGVDSVMCPKTDRLAAW
ARLASDLDPAKLEEMYTELPFSEVIETAPKFLDGTVRGRIVIPVTP*
Sbm MSNVQEWQQLANKELSRREKTVDSLVHQTAEGIAIKPLYTEADLDNL
SEQ ID EVTG`FLPGLPPYNRGPRATMYTAQPWFIRQYAGFSTAKESNAFYRRNL
NO: 61 AAGQICGLSVAFDLATHRGYDSDNPRVAGDVGKAGVAIDTVEDMKVL
FDQIPLD KMSVSMTMNGAV LPVLAFYIVAAEEQGVIPDKLTGT IQNDI
LKEYLCRNTYIYPPKRSIVIRIIADIIAWCSGNMPRFNTISISGYHMGEAGA
NCVQQVAFTLADGIEYIKAAISAGLKIDDFAPRLSFFFGIGMDLFMNVA
MLRAARYLWSEAVSGFGAQDPKSLALRTHccgsGwsufEQDPYNNVI
RTTIEALAATLGGTQSUITNAFDEALGLPTDFSARI ARNTQIIIQEESELC

LEASAREQS L1:DQG KRVI VG VN KY KLDHE DET Man:DNA/NI VRNEQIA
SLERIRATRDDAAVTAALNALTHAAQHNENLLAAAVNAARVRATLGE
IS DA-LEV-A FDRY-L VPS QC V TG VI AQS Y FIQS E KS ASEFDAIVAQTEQFLA
DNGRRPRILIAKNIGQDGHDRGAKVIASAYSDLGFDVDLSPNIFSTPEEIA
RLAVENDVHVVG AS S LAAGHKTLIPELVEALKKWGREDICVVAGGVIP
PQDYAFTAXRG V A AIYGPGTPMLDS VRDVLNLISQIIHD*
ygfD MINEATLAES IRRLRQGERATLAQ AMTLVES RHPRHQALSTQLLD AIM
SEQ ID PYCGNTLRLGVTGTPGAGKSTFLEAFGMLIAREGLKVAVIAVDPSSPVT
NO: 62 GGS ILGD KTRMNDLARAEAAFIRPVPS S GHLGGAS QRARELMLLCEAA
GYDV VIVETVGVGQSETEV ARM VDCFIS LQIAGGGDDLQGIKKGLME
VADLIVINKDDGDNHTNVAIARHMYES AI ,HILRRKY DEWQPRVLTCS
ALEKRGIDEIW HAIIDFKTALTASGRLQQVRQQQS VEWLRKQTEEEVL
NHLTANEDFDRYYRQTLLAVKNNTLS PRTGLRQLSEFIQTQYFD*
ygfG MS YQYVNVVIINKV AVM EN YGRKLN ALS KVFIDDL MQ ALS DLNRPEI
SEQ ID RCHLRAPSGS KVFSAGHDIHELPSGGRDPLS YDDPLRQITRMIQKFPKPI
NO: 63 IS MVEGSVWGGAFEMIMSS DLIIAASTSTFSMTPVNLGVPYNLVGIHNL
RDAG Fill V KIELIFT AS PITAQRAL AV GILINI-IV VE VEELEDET LQM AH1-I
ISEIKAPLAIAVIKEELRVLGEAHTMNSDEFERIQGNIRRAVYDSEDYQEG
MNAFLEKRKPNFVGH*
ygfH METQWTRMTANEAAE11QHNDMVAFSGFTPAGSPKALVF AI ARRANEQ
SEQ ID LIE AKKPYQIRLUM AS ISAAADD VLSDADAVSWRAPYQTS SGL RKKIN
NO: 64 QGAVS FVDLHLSEVAQMVNYGFFGDIDVAVIEAS ALAPDGRVWLTS GI
GNAPTWLLRAKKVIIELNHYHDPRVAELADIVIPGAPPRRNSVSIFHAM
DRVGTRYVQIIRKKIVAVVETNLPDAGNMLDKQNPMCQQIADNVVTF

LLQEMAHGRIPPEFLPLQSGVGNINNANTMARLGENPVIPPFMMYSEVL
QES VNI HELLEMKIS GAS AS S LTI S ADSLR DN YF AS RWLMEIS
NNPEIIRRI ,GVIALNVGI EFDIY Gil ANSTIIVAGVDLMNGIGGSGDPERN
AYLSIFMAPSIAKEGKIST VVPMCSFIVDHSEFIS VKVIITEQGIADLRGLS
PI ,QRART I DNC AI IPMY RD Y I A IRY 114,N APCK3 Ell III I DI SIR I DLL !RNLI
ATGSMI G*
mutA MS S TDQGTNPADTDDLTPTTLS LAGDFP KAT EEQWEREVEKVFNRGRPP
SEQ ID EKQLT FAECLKRLTVHTVDGIDIVPMYRP KD AP KKLGYPGVTPFTRGTT
NO: 65 VRNGDMDAWDVRALHEDPDEKFTRKAILEDLERGVTSLLLRVDPDAIA
PEHLDEVLSDVLLEMTKVEVFSRYDQGAAAEALMGVYERSDKPAKDLA
LNLGLDPIGFAALQGTEPDLTVLGDWVRRLAKFS PDS RAVTIDANVYHN
AGAGDVAELAWALATGAEYVRALVEQGFNATEAFDTINFRVTATHDQF
LTIARLRALREAWARIGEVFGVDEDKRGARQNAITSWRELTREDPYVNI
LRGSIATFS AS VGGAESITTLPFTQALGLPEDDFPLRIARNTGIVLAEEVNI
GRVNDPAGGSYYVESLTRTLADAAWKEFQEVEKLGGMS KAVMTEHVT
KVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFPTAPARKGLA
WHRDSEVFEQLMDRS TS VS ERPKVFLACLGTRRDFGGREGFS SPVWHIA
GIDTPQVEGGTTAEIVEAFKKS GAQVADLCS SAKIYAQQGLEVAKALKA
AGAKALYLS GAFKEFGDDAAEAEKLIDGRLYMGMDVVDTLS STLDILG
VAK
mutB VS TLPRFDS VDLGNAPVPADAAQRFEELAAKAGTEEAWETAEQIPVGTL
SEQ ID FNEDVYKDMDWLDTYAGIPPFVHGPYATMYAFRPWTIRQYAGFS TAKE
NO: 66 SNAFYRRNLAAGQKGLSVAFDLPTHRGYDS DNPRVAGDVGMAGVAIDS
IYDMRELFAGIPLDQMS VS MTMNGAVLPILALYVVTAEEQGVKPEQLA
GTIQNDILKEFMVRNTYIYPPQPS MRIISEIFAYTS ANMP KWNS IS IS GYH
MQEAGATADIEMAYTLADGVDYIRAGES VGLNVDQFAPRLSFFWGIGM
NFFMEVAKLRAARMLWAKLVHQFGPKNPKS MS LRTHS QTS GWS LT AQ
DVYNNVVRTCIEAMAAT QGHT QS LHTNS LDEAIALPTDFS ARIARNTQL
FLQQES GTTRVIDPWS GS AYVEELTWDLARKAWGHIQEVEKVGGMAK
AIEKGIPKMRIEEAAARTQARIDS GRQPLIGVNKYRLEHEPPLDVLKVDN
S TVLAEQKAKLVKLRAERDPEKVKAALDKITWAAANPDDKDPDRNLLK
LCID AGRAMATVGEMS D ALE KVFGRYT AQIRTIS GVYS KEVKNTPEVEE
ARELVEEFEQAEGRRPRILLAKMGQD GHDRGQKVIATAYADLGFDVDV
GPLFQTPEETARQAVEADVHVVGVS S LAGGHLTLVPALRKELDKLGRP
DILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLVKKLRAS LD A
GI:180421 MSNEDLFICIDHVAYACPDADEAS KYYQETFGWHELHREENPEQGVVEI

SEQ ID RVDDIDAVS ATLRERGVQLLYDEPKLGTGGNRINFMHPKS GKGVLIELT
NO: 67 QYPKN
mmdA MAENNNLKLASTMEGRVEQLAEQRQVIEAGGGERRVEKQHS QGKQTA
SEQ ID RERLNNLLDPHS FDEVGAFRKHRTTLFGMDKAVVPADGVVTGRGTILG
NO: 68 RPVHAAS QDFTVMGGS AGET QS TKVVETMEQALLTGTPFLFFYDS GGA
RIQEGIDS LS GYGKMFFANVKLS GVVPQIAIIAGPCAGGASYSPALTDFII
MTKKAHMFITGPQVIKS VT GEDVTADELGGAE AHMAIS GNIHFVAEDD
DAAELIAKKLLS FLPQNNTEEASFVNPNNDVSPNTELRDIVPIDGKKGYD
VRDVIAKIVDWGDYLEVKAGYATNLVTAFARVNGRS VGIVANQPS VMS
GCLDINASDKAAEFVNFCDS FNIPLVQLVDVPGFLPGVQQEYGGIIRHGA

KMLYAYSEATVPKITVVLRKAYGGSYLAMCNRDLGADAVYAWPS AEI
AVMGAEGAANVIFRKEIKAADDPDAMRAEKIEEYQNAFNTPYVAAARG
QVDDVIDPADTRRKIASALEMYATKRQTRPAKKHGNFPC
PFREUD MSPREIEVSEPREVGITELVLRDAHQSLMATRMAMEDMVGACADIDAA

SEQ ID LLGYRHYNDEVVDRFVDKSAENGMDVFRVFDAMNDPRNMAHAMAAV
NO: 69 KKAGKHAQGTICYTISPVHTVEGYVKLAGQLLDMGADSIALKDMAALL
KPQPAYDIIKAIKDTYGQKTQINLHCHSTTGVTEVSLMKAIEAGVDVVD
TAISSMSLGPGHNPTESVAEMLEGTGYTTNLDYDRLHKIRDHFKAIRPKY
KKFESKTLVDTSIFKSQIPGGMLSNMESQLRAQGAEDKMDEVMAEVPR
VRKAAGFPPLVTPSSQIVGTQAVFNVMMGEYKRMTGEFADIMLGYYGA
SPADRDPKVVKLAEEQSGKKPITQRPADLLPPEWEEQSKEAAALKGFNG
TDEDVLTYALFPQVAPVFFEHRAEGPHSVALTDAQLKAEAEGDEKSLAV
AGPVTYNVNVGGTVREVTVQQA
Bccp MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPRAAGGA
SEQ ID GAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEIN
NO: 70 APTDGKVEKVLVKERDAVQGGQGLIKIG
[0284] In some embodiments, the genetically engineered bacteria encode one or more polypeptide sequences of Table 7 (SEQ ID NO: 46-SEQ ID NO: 70, and SEQ ID

NO: 20) or a functional fragment or variant thereof. In some embodiments, genetically engineered bacteria comprise a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%
homologous to the polypeptide sequence of one or more polypeptide sequence of Table 7 (SEQ
ID NO:
46-SEQ ID NO: 70, and SEQ ID NO: 20) or a functional fragment thereof.
[0285] In one embodiment, the bacterial cell comprises a non-native or heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a non-native or heterologous propionate gene cassette operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a propionate gene cassette, as well as at least one copy of a propionate gene cassette from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette.

-150-[0286] Multiple distinct propionate gene cassettes are known in the art. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the propionate gene cassette is derived from an organism of the genus or species that includes, but is not limited to, Clostridium propionicum, Megasphaera elsdenii, or Prevotella ruminicola.
[0287] In one embodiment, the propionate gene cassette has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the propionate gene cassette has been codon-optimized for use in Escherichia coli. In another embodiment, the propionate gene cassette has been codon-optimized for use in Lactococcus. When the propionate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more propionate than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat autoimmune disease, such as IBD.
[0288] The present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme. As used herein, the term "functional fragment thereof' or "functional variant thereof' relates to an element having qualitative biological activity in common with the wild-type enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated propionate biosynthesis enzyme is one which retains essentially the same ability to synthesize propionate as the propionate biosynthesis enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having propionate biosynthesis enzyme activity may be truncated at the N-terminus or C-terminus, and the retention of propionate biosynthesis enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant. In another embodiment, the engineered bacterial

-151-cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment.
[0289] As used herein, the term "percent (%) sequence identity" or "percent (%) identity," also including "homology," is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2,482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl.
Acad.
Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).
[0290] The present disclosure encompasses propionate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A
conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid.
Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S
and T.
Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid

-152-with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).
[0291] In some embodiments, a propionate biosynthesis enzyme is mutagenized;
mutants exhibiting increased activity are selected; and the mutagenized gene encoding the propionate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.
[0292] In one embodiment, the propionate biosynthesis gene cassette is from Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium propionicum.
In another embodiment, the propionate biosynthesis gene cassette is from a Megasphaera spp. In one embodiment, the Megasphaera spp. is Megasphaera elsdenii. In another embodiment, the propionate biosynthesis gene cassette is from Prevotella spp.
In one embodiment, the Prevotella spp. is Prevotella ruminicola. Other propionate biosynthesis gene cassettes are well-known to one of ordinary skill in the art.
[0293] In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, e0, acrB, and acrC. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and 1pd, and optionally further comprise tesB. The genes may be codon-optimized, and translational and transcriptional elements may be added.
[0294] In one embodiment, the pct gene has at least about 80% identity with SEQ
ID NO: 21. In another embodiment, the pct gene has at least about 85% identity with SEQ ID NO: 21. In one embodiment, the pct gene has at least about 90% identity with SEQ ID NO: 21. In one embodiment, the pct gene has at least about 95% identity with SEQ ID NO: 21. In another embodiment, the pct gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. Accordingly, in one embodiment, the pct gene has at least about 80%, 821%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 921%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In another embodiment, the pct gene comprises the sequence of SEQ ID NO: 21. In yet another embodiment the pct gene consists of the sequence of SEQ ID NO: 21.

-153-[0295] In one embodiment, the lcdA gene has at least about 80% identity with SEQ ID NO: 22. In another embodiment, the lcdA gene has at least about 85%
identity with SEQ ID NO: 22. In one embodiment, the lcdA gene has at least about 90%
identity with SEQ ID NO: 22. In one embodiment, the lcdA gene has at least about 95%
identity with SEQ ID NO: 22. In another embodiment, the lcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 822%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 922%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
22. In another embodiment, the lcdA gene comprises the sequence of SEQ ID NO:
22. In yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO:
22.
[0296] In one embodiment, the lcdB gene has at least about 80% identity with SEQ ID NO: 23. In another embodiment, the lcdB gene has at least about 85%
identity with SEQ ID NO: 23. In one embodiment, the lcdB gene has at least about 90%
identity with SEQ ID NO: 23. In one embodiment, the lcdB gene has at least about 95%
identity with SEQ ID NO: 23. In another embodiment, the lcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. Accordingly, in one embodiment, the lcdB gene has at least about 80%, 81%, 82%, 823%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 923%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
23. In another embodiment, the lcdB gene comprises the sequence of SEQ ID NO:
23. In yet another embodiment the lcdB gene consists of the sequence of SEQ ID NO:
23.
[0297] In one embodiment, the lcdC gene has at least about 80% identity with SEQ ID NO: 24. In another embodiment, the lcdC gene has at least about 85%
identity with SEQ ID NO: 24. In one embodiment, the lcdC gene has at least about 90%
identity with SEQ ID NO: 24. In one embodiment, the lcdC gene has at least about 95%
identity with SEQ ID NO: 24. In another embodiment, the lcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 24. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 824%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 924%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
24. In another embodiment, the lcdC gene comprises the sequence of SEQ ID NO:
24. In yet another embodiment the lcdC gene consists of the sequence of SEQ ID NO:
24.
[0298] In one embodiment, the e0 gene has at least about 80% identity with SEQ

ID NO: 25. In another embodiment, the etfA gene has at least about 825%
identity with

-154-SEQ ID NO: 25. In one embodiment, the e0 gene has at least about 90% identity with SEQ ID NO: 25. In one embodiment, the e0 gene has at least about 925% identity with SEQ ID NO: 25. In another embodiment, the e0 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. Accordingly, in one embodiment, the etfA
gene has at least about 80%, 81%, 82%, 83%, 84%, 825%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 925%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25.
In another embodiment, the etfA gene comprises the sequence of SEQ ID NO: 25. In yet another embodiment the e0 gene consists of the sequence of SEQ ID NO: 25.
[0299] In one embodiment, the acrB gene has at least about 80% identity with SEQ ID NO: 26. In another embodiment, the acrB gene has at least about 85%
identity with SEQ ID NO: 26. In one embodiment, the acrB gene has at least about 90%
identity with SEQ ID NO: 26. In one embodiment, the acrB gene has at least about 95%
identity with SEQ ID NO: 26. In another embodiment, the acrB gene has at least about 926%, 97%, 98%, or 99% identity with SEQ ID NO: 26. Accordingly, in one embodiment, the acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 826%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 926%, 97%, 98%, or 99% identity with SEQ ID NO:
26. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO:
26.
In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO:
26.
[0300] In one embodiment, the acrC gene has at least about 80% identity with SEQ ID NO: 27. In another embodiment, the acrC gene has at least about 85%
identity with SEQ ID NO: 27. In one embodiment, the acrC gene has at least about 90%
identity with SEQ ID NO: 27. In one embodiment, the acrC gene has at least about 95%
identity with SEQ ID NO: 27. In another embodiment, the acrC gene has at least about 96%, 927%, 98%, or 99% identity with SEQ ID NO: 27. Accordingly, in one embodiment, the acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 827%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 927%, 98%, or 99% identity with SEQ ID NO:
27. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO:
27.
In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO:
27.
[0301] In one embodiment, the thrAfbr gene has at least about 280% identity with SEQ ID NO: 28. In another embodiment, the thrAfbr gene has at least about 285%

identity with SEQ ID NO: 28. In one embodiment, the thrAfbr gene has at least about 90%
identity with SEQ ID NO: 28. In one embodiment, the thrAfbr gene has at least about 95%

-155-identity with SEQ ID NO: 28. In another embodiment, the thrgbr gene has at least about 96%, 97%, 928%, or 99% identity with SEQ ID NO: 28. Accordingly, in one embodiment, the thrgbr gene has at least about 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 2828%, 289%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 928%, or 99%
identity with SEQ ID NO: 28. In another embodiment, the thrgbr gene comprises the sequence of SEQ ID NO: 28. In yet another embodiment the thrgbr gene consists of the sequence of SEQ ID NO: 28.
[0302] In one embodiment, the thrB gene has at least about 80% identity with SEQ ID NO: 29. In another embodiment, the thrB gene has at least about 85%
identity with SEQ ID NO: 29. In one embodiment, the thrB gene has at least about 290%
identity with SEQ ID NO: 29. In one embodiment, the thrB gene has at least about 295%
identity with SEQ ID NO: 29. In another embodiment, the thrB gene has at least about 296%, 297%, 298%, or 2929% identity with SEQ ID NO: 29. Accordingly, in one embodiment, the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 829%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, or 2929% identity with SEQ ID NO: 29. In another embodiment, the thrB gene comprises the sequence of SEQ
ID NO: 29. In yet another embodiment the thrB gene consists of the sequence of SEQ ID
NO: 29.
[0303] In one embodiment, the thrC gene has at least about 80% identity with SEQ ID NO: 30. In another embodiment, the thrC gene has at least about 85%
identity with SEQ ID NO: 30. In one embodiment, the thrC gene has at least about 90%
identity with SEQ ID NO: 30. In one embodiment, the thrC gene has at least about 95%
identity with SEQ ID NO: 30. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 30. Accordingly, in one embodiment, the thrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
30. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO:
30. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO:
30.
[0304] In one embodiment, the i/vAfbr gene has at least about 80% identity with SEQ ID NO: 31. In another embodiment, the i/vAfbr gene has at least about 85%
identity with SEQ ID NO: 31. In one embodiment, the i/vAfbr gene has at least about 90%
identity with SEQ ID NO: 31. In one embodiment, the i/vAfbr gene has at least about 95%
identity

-156-with SEQ ID NO: 31. In another embodiment, the i/vAfbr gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 31. Accordingly, in one embodiment, the i/vAfbr gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
31. In another embodiment, the i/vAfbr gene comprises the sequence of SEQ ID
NO: 31.
In yet another embodiment the i/vAfbr gene consists of the sequence of SEQ ID
NO: 31.
[0305] In one embodiment, the aceE gene has at least about 80% identity with SEQ ID NO: 32. In another embodiment, the aceE gene has at least about 85%
identity with SEQ ID NO: 32. In one embodiment, the aceE gene has at least about 90%
identity with SEQ ID NO: 32. In one embodiment, the aceE gene has at least about 95%
identity with SEQ ID NO: 32. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 32. Accordingly, in one embodiment, the aceE gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
32. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO:
32.
In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO:
32.
[0306] In one embodiment, the aceF gene has at least about 80% identity with SEQ ID NO: 33. In another embodiment, the aceF gene has at least about 85%
identity with SEQ ID NO: 33. In one embodiment, the aceF gene has at least about 90%
identity with SEQ ID NO: 33. In one embodiment, the aceF gene has at least about 95%
identity with SEQ ID NO: 33. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 33. Accordingly, in one embodiment, the aceF gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
33. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO:
33.
In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO:
33.
[0307] In one embodiment, the 1pd gene has at least about 80% identity with SEQ
ID NO: 34. In another embodiment, the 1pd gene has at least about 85% identity with SEQ ID NO: 34. In one embodiment, the 1pd gene has at least about 90% identity with SEQ ID NO: 34. In one embodiment, the 1pd gene has at least about 95% identity with SEQ ID NO: 34. In another embodiment, the 1pd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 34. Accordingly, in one embodiment, the 1pd gene has

-157-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: 34. In another embodiment, the 1pd gene comprises the sequence of SEQ ID NO: 34. In yet another embodiment the 1pd gene consists of the sequence of SEQ ID NO: 34.
[0308] In one embodiment, the tesB gene has at least about 80% identity with SEQ
ID NO: 10. In another embodiment, the tesB gene has at least about 85%
identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90%
identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95%
identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the tesB
gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 10.
[0309] In one embodiment, the actd gene has at least about 80% identity with SEQ ID NO: 35. In another embodiment, the actd gene has at least about 85%
identity with SEQ ID NO: 35. In one embodiment, the actd gene has at least about 90%
identity with SEQ ID NO: 35. In one embodiment, the actd gene has at least about 95%
identity with SEQ ID NO: 35. In another embodiment, the actd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 35. Accordingly, in one embodiment, the actd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
35. In another embodiment, the actd gene comprises the sequence of SEQ ID NO:
35. In yet another embodiment the actd gene consists of the sequence of SEQ ID NO:
35.
[0310] In one embodiment, the sbm gene has at least about 80% identity with SEQ
ID NO: 36. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 36. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 36. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 36. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 36Ø Accordingly, in one embodiment, the sbm gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 36. In

-158-another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 36. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 36.
[0311] In one embodiment, the ygfD gene has at least about 80% identity with SEQ ID NO: 37. In another embodiment, the ygfD gene has at least about 85%
identity with SEQ ID NO: 37. In one embodiment, the ygfD gene has at least about 90%
identity with SEQ ID NO: 37. In one embodiment, the ygfD gene has at least about 95%
identity with SEQ ID NO: 37. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37. Accordingly, in one embodiment, the ygfD gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
37. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO:
37.
In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO:
37.
[0312] In one embodiment, the ygfG gene has at least about 80% identity with SEQ ID NO: 38. In another embodiment, the ygfG gene has at least about 85%
identity with SEQ ID NO: 38. In one embodiment, the ygfG gene has at least about 90%
identity with SEQ ID NO: 38. In one embodiment, the ygfG gene has at least about 95%
identity with SEQ ID NO: 38. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38.. Accordingly, in one embodiment, the ygfG gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
38. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO:
38.
In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO:
38.
[0313] In one embodiment, the ygfH gene has at least about 80% identity with SEQ ID NO: 39. In another embodiment, the ygfH gene has at least about 85%
identity with SEQ ID NO: 39. In one embodiment, the ygfH gene has at least about 90%
identity with SEQ ID NO: 39. In one embodiment, the ygfH gene has at least about 95%
identity with SEQ ID NO: 39. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. .Accordingly, in one embodiment, the ygfH gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
39. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO:
39.
In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO:
39.

-159-[0314] In one embodiment, the mutA gene has at least about 80% identity with SEQ ID NO: 40. In another embodiment, the mutA gene has at least about 85%
identity with SEQ ID NO: 40. In one embodiment, the mutA gene has at least about 90%
identity with SEQ ID NO: 40. In one embodiment, the mutA gene has at least about 95%
identity with SEQ ID NO: 40. In another embodiment, the mutA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. .Accordingly, in one embodiment, the mutA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
40. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO:
40.
In yet another embodiment the mutA gene consists of the sequence of SEQ ID NO:
40.
[0315] In one embodiment, the mutB gene has at least about 80% identity with SEQ ID NO: 41. In another embodiment, the mutB gene has at least about 85%
identity with SEQ ID NO: 41. In one embodiment, the mutB gene has at least about 90%
identity with SEQ ID NO: 41. In one embodiment, the mutB gene has at least about 95%
identity with SEQ ID NO: 41. In another embodiment, the mutB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. .Accordingly, in one embodiment, the mutB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:
41. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO:
41.
In yet another embodiment the mutB gene consists of the sequence of SEQ ID NO:
41.
[0316] In one embodiment, the GI 18042134 gene has at least about 80% identity with SEQ ID NO: 42. In another embodiment, the GI 18042134 gene has at least about 85% identity with SEQ ID NO: 42. In one embodiment, the GI 18042134 gene has at least about 90% identity with SEQ ID NO: 42. In one embodiment, the GI

gene has at least about 95% identity with SEQ ID NO: 42. In another embodiment, the GI 18042134 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID
NO:
42..Accordingly, in one embodiment, the GI 18042134 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42. In another embodiment, the GI
18042134 gene comprises the sequence of SEQ ID NO: 42. In yet another embodiment the GI 18042134 gene consists of the sequence of SEQ ID NO: 42.

-160-[0317] In one embodiment, the mmdA gene has at least about 80% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene has at least about 85%
identity with SEQ ID NO: 43. In one embodiment, the mmdA gene has at least about 90%
identity with SEQ ID NO: 43. In one embodiment, the mmdA gene has at least about 95% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43..Accordingly, in one embodiment, the mmdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene comprises the sequence of SEQ ID NO: 43. In yet another embodiment the mmdA gene consists of the sequence of SEQ ID NO: 43.
[0318] In one embodiment, the PFREUD 188870 gene has at least about 80%
identity with SEQ ID NO: 44. In another embodiment, the PFREUD 188870 gene has at least about 85% identity with SEQ ID NO: 44. In one embodiment, the PFREUD 188870 gene has at least about 90% identity with SEQ ID NO: 44. In one embodiment, the PFREUD 188870 gene has at least about 95% identity with SEQ ID

NO: 44. In another embodiment, the PFREUD 188870 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44..Accordingly, in one embodiment, the PFREUD 188870 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ
ID NO: 44. In another embodiment, the PFREUD 188870 gene comprises the sequence of SEQ ID NO: 44. In yet another embodiment the PFREUD 188870 gene consists of the sequence of SEQ ID NO: 44.
[0319] In one embodiment, the Bccp gene has at least about 80% identity with SEQ ID NO: 45. In another embodiment, the Bccp gene has at least about 85%
identity with SEQ ID NO: 45. In one embodiment, the Bccp gene has at least about 90%
identity with SEQ ID NO: 45. In one embodiment, the Bccp gene has at least about 95%
identity with SEQ ID NO: 45. In another embodiment, the Bccp gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. .Accordingly, in one embodiment, the Bccp gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

-161-45. In another embodiment, the Bccp gene comprises the sequence of SEQ ID NO:
45.
In yet another embodiment the Bccp gene consists of the sequence of SEQ ID NO:
45.
[0320] In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%
identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ
ID NO: 46 through SEQ ID NO: 70. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70.
Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 46 through SEQ
ID
NO: 70. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 46 through SEQ ID NO: 70.
In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or or more of SEQ
ID NO: 46 through SEQ ID NO: 70.
[0321] In some embodiments, one or more of the propionate biosynthesis genes is a synthetic propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is an E. coli propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C.
glutamicum propionate biosynthesis gene. In some embodiments, one or more of the propionate

-162-biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a R.
sphaeroides propionate biosynthesis gene. The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate.
[0322] To improve acetate production, while maintaining high levels of propionate production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Deletions which may be introduced therefore include deletion of adhE, ldh, and frd.
Thus, in certain embodiments, the genetically engineered bacteria comprise one or more propionate cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0323] In some embodiments, the genetically engineered bacteria comprise one or more propionate cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE
gene.
[0324] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous frdA
gene. In some embodiments, the genetically engineered bacteria comprise one or more

-163-gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA and rdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
[0325] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous adhE
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH
gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA
gene. In some embodiments, the genetically engineered bacteria comprise one or more

-164-gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH
gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA
genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous frdA and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0326] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-

-165-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0327] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0328] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of propionate production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for propionate production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for propionate synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes.
[0329] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s)

-166-encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
[0330] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE

-167-gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta and ldhA genes.
[0331] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH
propionate cassette(s) and further comprise a mutation in the endogenous pta and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one

-168-or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE
genes.
[0332] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0333] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria

-169-produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0334] In some embodiments, the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate. In some embodiments, one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production. In some embodiments, the local production of propionate reduces food intake and improves gut barrier function and reduces inflammation In some embodiments, the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0335] In one embodiment, the propionate gene cassette is directly operably linked to a first promoter. In another embodiment, the propionate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the propionate gene cassette in nature.
[0336] In some embodiments, the propionate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the propionate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the propionate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.
[0337] The propionate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the propionate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the propionate gene cassette is

-170-located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located on a plasmid in the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.
[0338] In some embodiments, the propionate gene cassette is expressed on a low-copy plasmid. In some embodiments, the propionate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of propionate.
Tryptophan and Tryptophan Metabolism Kynurenine [0339] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine. Kynurenine is a metabolite produced in the first, rate-limiting step of tryptophan catabolism. This step involves the conversion of tryptophan to kynurenine, and may be catalyzed by the ubiquitously-expressed enzyme indoleamine 2,3-dioxygenase (IDO-1), or by tryptophan dioxygenase (TDO), an enzyme which is primarily localized to the liver (Alvarado et al., 2015). Biopsies from human patients with IBD show elevated levels of IDO- 1 expression compared to biopsies from healthy individuals, particularly near sites of ulceration (Ferdinande et al., 2008; Wolf et al., 2004). IDO-1 enzyme expression is similarly upregulated in trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced mouse models of IBD; inhibition of IDO- 1 significantly augments the inflammatory response caused by each inducer (Ciorba et al., 2010; Gurtner et al., 2003;
Matteoli et al., 2010). Kynurenine has also been shown to directly induce apoptosis in neutrophils (El-Zaatari et al., 2014). Together, these observations suggest that IDO-1 and kynurenine play a role in limiting inflammation. The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise a gene or gene cassette for producing a

-171-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 anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions.
[0340] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD
patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene, genes, or gene cassettes for producing kynurenic acid. In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions Tryptophan, Tryptophan Metabolism, and Tryptophan Metabolites Tryptophan and the Kynurenine Pathway

-172-[0341] Tryptophan (TRP) is an essential amino acid that, after consumption, is either incorporated into proteins via new protein synthesis, or converted a number of biologically active metabolites with a number of differing roles in health and disease (Perez-De La Cruz et al., 2007 Kynurenine Pathway and Disease: An Overview;
CNS&Neurological Disorders -Drug Targets 2007, 6,398-410). Along one arm of tryptophan catabolism, trytophan is converted to the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) by tryptophan hydroxylase. Serotonin can further be converted into the hormone melatonin. A large share of tryptophan, however, is metabolized to a number of bioactive metabolites, collectively called kynurenines, along a second arm called the kynurenine pathway (KP). In the first step of catabolism, TRP is converted to Kynurenine, (KYN), which has well-documented immune suppressive functions in several types of immune cells, and has recently been shown to be an activating ligand for the arylcarbon receptor (AhR; also known as dioxin receptor). KYN was initially shown in the cancer setting as an endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival. In the gut, kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism.
[0342] More recently, additional tryptophan metabolites, collectively termed "indoles", herein, including for example, indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc. which are generated by the microbiota, some by the human host, some from the diet, which are also able to function as AhR agonists, see e.g., Table 8 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.
[0343] Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in additiona to kynurenine, tryptophan metabolites L-kynurenine, 6-formylindolcarbazole (FICZ, a photoproduct of TRP), and KYNA are have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-KB
subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR-ARNT

-173-associated transcription of IL-6, which induced autocrine activation of IDO1 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.
[0344] In the gut, tryptophan may also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell respononse and promotion of Treg cells.
[0345] The rate-limiting conversion of TRP to KYN may be mediated by either of two forms of indoleamine 2, 3-dioxygenase (IDO) or by tryptophan 2,3-dioxygenase (TDO). One characteristic of TRP metabolism is that the rate-limiting step of the catalysis from TRP to KYN is generated by both the hepatic enzyme tryptophan 2,3-dioxygenase (TDO) and the ubiquitous expressed enzyme ID01. TDO is essential for homeostasis of TRP concentrations in organisms and has a lower affinity to TRP than ID01. Its expression is activated mainly by increased plasma TRP concentrations but can also be activated by glucocorticoids and glucagon. The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut (as shown in the figures and the examples, 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. Along one pathway, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA) which can antagonize glutamate receptors and can also bind AHR and also GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-aspartate (NMDA)-receptors, and others. Along a third pathway of the KP, KYN can be converted to anthranilic acid (AA) and further downstream quinolinic acid (QUIN), which is a glutamate receptor agonist and has a neurotoxic role.
[0346] Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of a number of diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning trypophan and tryptophan

-174-metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites. and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.
Other Indole Tryptophan Metabolites [0347] 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 Reports 5:12689).
[01] 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).
[0348] Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states.
Murine models have demonstrated improved intestinal inflammation states following administration of 11-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.
[0349] 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

-175-disclosure. Thus, in some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes for the production of one or more metabolites listed in Table 8.
Table 8. Indole Tryptophan Metabolites Origin Compound Exogenous 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Dietary Indole-3-carbinol (I3C) Dietary Indole-3-acetonitrile (I3ACN) Dietary 3.3'-Diindolylmethane (DIM) Dietary 2-(indo1-3-ylmethyl)-3.3'-diindolylmethane (Ltr-1) Dietary Indolo(3,2-b)carbazole (ICZ) Dietary 2-(1'H-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 Indoxy1-3-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

-176-[0350] 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 (Nr1i2-/-) mice showed a distinctly "leaky' gut physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, August 21, 2014). In particular, indole 3-propionic acid (IPA), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo.
[0351] As a result of PXR agonism, indole levels e.g., produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR
regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. I.e., low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinaly barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health.
[0352] In other embodiments, IPA producing circuits comprise enzymes depicted and described in the figures and elsewhere herein. Thus, in some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more enzymes selected from TrpDH: tryptophan dehydrogenase (e.g., from from Nostoc punctiforme NIES-2108); FldHl/F1dH2: 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);
AcuI: acrylyl-CoA reductase (e.g., from Rhodobacter sphaeroides); 1pdC: Indole-3-pyruvate decarboxylase (e.g., from Enterobacter cloacae); ladl: Indole-3-acetaldehyde dehydrogenase (e.g., from Ustilago maydis); and Tdc: Tryptophan decarboxylase (e.g., from Catharanthus roseus or from Clostridium sporogenes). In some embodiments, the engineered bacteria comprise gene sequence(s) and/or gene cassette(s) for the production of one or more of the following: indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA).
[0353] Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indo1-3-

-177-yl)pyruvate (IPyA), NH3, NAD(P)H and H. Indole-3-lactate dehydrogenase ((EC
1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) converts (indo1-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-propionyl-CoA:indole-3-lactate CoA transferase (F1dA ) converts indole-3-lactate (ILA) and indo1-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA.
Indole-3-acrylyl-CoA reductase (F1dD ) and acrylyl-CoA reductase (AcuI) 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. Indole-3-pyruvate decarboxylase (1pdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d) ladl:
Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA).
[0354] 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 1;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-acrylate to indole-3-propionate (O'Neill and DeMoss, Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys.
1968 Sep 20;127(1):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).
[0355] L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g., Clostridium sporogenes or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indo1-3y1)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g., Clostridium

-178-sporogenes orLactobacillus casei) converts (indo1-3y1) pyruvate and NADH and H+ to indole-3 lactate and NAD+.
[0356] In some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes selected from tryptophan transaminase (e.g., from C.
sporogenes) and/or indole-3-lactate dehydrogenase (e.g., from C. sporogenes), and/or indole-3-pyruvate aminotransferase (e.g., from Lactococcus lactis). In other embodiments, such enzymes encoded by the bacteria are from Lactobacillus casei and/or Lactobacillus helveticus.
[0357] In other embodiments, IPA producing circuits comprise enzymes depicted and described in FIG. 47 and FIG. 48 and elsewhere herein.
[0358] In some embodiments, the bacteria comprise gene sequence for producing one or more tryptophan metabolites, e.g., "indoles". In some embodiments, the bacteria comprise gene sequence for producing and indole selected from indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ. In some embodiments, the bacteria comprise gene sequence for producing an indole that functions as an AhR agonist, see e.g., Table 8..
[0359] In some embodiments, the bacteria comprise any one or more of the circuits described and depicted in the figures and examples.
Methoxyindole pathway, Serotonin and Melatonin [0360] 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). Subsequently, 5-HTP undergoes decarboxylation to serotonin.
[0361] 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

-179-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.ce11.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis).
[0362] Modulation of tryptophan metabolism, especially serotonin synthesis is considered a novel potential strategy the treatment of gastrointestinal (GI) disorders, including IBD.
[0363] In some embodiments, the engineered bacteria comprise gene sequence encoding one or more tryptophan hydroxylase genes (Tphl or Tph2). In some embodiments, the engineered bacteria further comprise gene sequence for decarboxylating 5-HTP. In some embodiments, the engineered bacteria comprise gene sequence for the production of 5-hydroxytryptophan (5-HTP). In some embodiments, the engineered bacteria comprise gene sequence for the production of seratonin.
[0364] In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the gut, e.g., decrease or increase serotonin levels, e.g, in the gut and in the circulation. 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, otherwise ameliorate symptoms of A gastrointestinal disorder or inflammatory 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 metabolic diseases.
[0365] In some embodiments, the genetically engineered bacteria comprise gene sequence encoding tryptophan hydroxylase (TpH (land/or2)) and/or 1-amino acid decarboxylase, e.g. for the treatment of constipation-associated metablic disorders. In

-180-some embodiments, the genetically engineered bacteria comprise genetic 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.
[0366] 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.
[0367] 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/s12154-011-0064-8.
[0368] In a non-limiting example, genetically engineered bacteria convert tryptophan and/or serotonin to melatonin by, e.g., tryptophan hydroxylase (TPH), hydroxyl-0-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic ¨
amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial equivalents.
Exemplary Tryptophan and Tryptophan Metabolite Circuits Decreasing Exogenous Tryptophan [0369] 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

-181-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.
[0370] 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).
[0371] 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. 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.
[0372] 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.
[0373] 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

-182-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.
[0374] 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, forty-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.
[0375] 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 8.
[0376] 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 comprise 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 gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase

-183-(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.
[0377] 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. In some embodiments, expression of the gene sequences(s) is driven by an inducible promoter, described in more detail herein. In some embodiments, the expression of the gene sequences(s) is driven by a constitutive promoter.
Increasing Kynurenine [0378] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine.
[0379] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprise one or more gene sequences for converting tryptophan to kynurenine.
In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise 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.
[0380] 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 the figures and described elsewhere herein. In one embodiment, the genetically

-184-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 IDO1 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)

-185-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.
[0381] 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.
[0382] 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 the figures 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.
[0383] 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 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 and others described herein. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a

-186-constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0384] 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.
[0385] In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein.
[0386] In any of the embodiments described above and elsewhere herein, 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, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the

-187-described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following:
(1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.
Increasing Tryptophan [0387] 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 the figures.
[0388] 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.
[0389] 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

-188-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.
[0390] In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant 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.
[0391] In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 10 [0392] 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.
[0393] 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 10.
[0394] 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

-189-embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.
[0395] In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan, 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. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0396] Table 9A and 9B lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure.
Table 9A. Tryptophan Synthesis Cassette Sequences Description Sequence Tet-regulated taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaaggctggctct Tryptophan gcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct operon tctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata SEQ ID NO:
atgcattctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtagg ccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa aaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacg ggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagac a tcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagt g aactctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactctcgaactgct aacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtggggatcgtccggcaacg ctgctgctggaatccgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactg gataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttcccgcctgtca gtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgctttccgcttattacagaatctgttga atgtaccgaaggaagaacgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaa atttaccgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgac catcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgc tcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtttccgtgccgcatat gcgttgtgaatgtaaccagagcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccg gagaaattttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgct gaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccctgtttggcgcgtcgccggaa agttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtc

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Eumr5v5ERro5Ear5ovv5Eo5oovE55p5p5p5oRro55ool5ov55551515115.roarol :ON m Oas Bpo5o5pr5ooaruar5o5ovip5o55Eu5o5parup5praolopaoarEERrarouRro5Tr .. actil DDIDDDIVDDIIDOVVDDDIDVDDDDIDDDILLII
DDDDIVDDDVDIDDIVDDDOVVDVDDVVIIVVVDIVDDDVDDDID
VVVIVDDODDDDDVDDVDDOODDDIDDDVDDDDDODDVVDDVVD
DDIIDDVVOLLIVDDDDVDDODDODDIVVVDVDDVIDVDIDDIDI
DODVVOIDDDIDLLIDIIDIDIVILLIDDLLIDDDDDIDVDVVVDD
IDVDIDDOVVVDDVVVVIVVVDIVDDDVDDDIDVVDDDVIDVDV
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o55aro5aro5o555.roparo5v5vpmEar5o5aro5u5oo5uop55E5Traumm5mo5510 :ON m Oas 5oaruo5uo5E5ERro5ooarEE5E15551BE5o55.ruar5uo5o15oTrEEE5o5EBB5oaruuo5Tr Dd.11 Eu1555E5aro55o55pro5oarol5E5Ear5oupo5oop5515m5o5p E155E5BoB5oaruuo5o5Truoo5u.ro5pw5Eu5voo55Tro5po5o5vEB5Troo5o15Tru5o 55o5o15oo5m5EaTr0005oo5ar5o55EuE155EuarB5Bo5ararupurar515oarEEE5Eu 55oararE55o55m551Truo5E55.roaroarlooaroapo55Bur5Eap5oar5uruovio5E
5EEBEEE5o55ar5Troularaoo5B5oTruar5oo5o5ararivom5EaTr551E555o55o5uo uo515515m55o5o5oRrov15555015o5o5BoarEE5oo5llaoo5p5155prE55ool5E1 Em5155llrup5o55p5oo5ooTro5o55ooarupauroar555p515oRrop5ppro5ooarE
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EarEBE55oo5m5v5o55m155ar5Traralvo5E5o15o5uum55aropolTruE5o555o55 uo155o15vio55555arpo5Eu5o15BE5o55.roTrou55opo5Bvo55lluivuoo5p5Euo551 5o5Bo5ooaropoprE55oo5m5B55oo5Eu5o5u5oo515155000055poppp5v5p515 9L
5ooTruo5E5Taar5o55po5arallumpar5m5o55oopuTroaruo5oarmr515515armr :ON m Oas o155Truo5uo5o5B5uoTr5uo55paruari5ouppopr5ovvEv5op5p5purar5p55Tr ad.11 upp5ou 5E55Earo5TroTro5o5oaroo5llup5o5o5pri5p5o50005uumul5opuraar5oo5EE5 5o15m5oop5pllapool5E15o55p5155oo5u.ro515oaroo5ov155arEEE55155p5o55 op5oov5151vo5parar5olov5o55Tro5o5oarmvp55E155o5o55o55ario5m55o5 ar5o15o155Eauo5Eu5oo5BEEB5m5vio5o5oul5EuE5oaro5155o5EEB5ar5555vw E5m5po5o5oarpo5aro5poo5ar5opTaaroo5o5p5E5155115115o5000pi5Eloaro5 v515arpopuB5oar5B5ERroaropTr5oo5o15ario5oo5m55ooararo5mro5aro5510 ar5vEl50005opv55155p5v5pvarE5pplo5E5EuEvoTr5oom5o5v5E55prE5ow 099I0/LIOZSI1IIDd tttccagacgctgcgcgcatattaa trpB
atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcg SEQ ID NO:
ccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaa actatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatc tgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcga agcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgcca gcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttc cggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatc cttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagaga aggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatc aacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgc accgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaa attgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagc actggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaa gggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaa aaagagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaa gcacgaggggaaatctga trpA
atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc SEQ ID NO:
ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagtt aggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcg ggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggcc ttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcga ttcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacac ctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcg aagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccg cgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagc cagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa Table 9B. Tryptophan Synthesis Polypeptide Sequences Description Sequence TrpE MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLL
SEQ ID NO: 75 LES ADIDS KDDLKSLLLVDS ALRITALSDTVTIQALSGN
GEALLTLLDNALPAGVENEQSPNCRVLRFPPVSPLLDE
DARLCS LS VFDAFRLLQNLLNVPKEEREAMFFGGLFS
YDLVAGFENLPQLSAENSCPDFCFYLAETLMVIDHQK
KS TRIQASLFAPNEEEKQRLT ARLNELRQQLTEAAPPL
PVVSVPHMRCECNQSDEEFGGVVRLLQKAIRAGEIFQ
VVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDND
FTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGS
LDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARI
CTPGSRYVADLTKVDRYSYVMHLVSRVVGELRHDLD
ALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGS
YGGAVGYFTAHGDLDTCIVIRSALVENGIATVQAGAG
VVLDSVPQSEADETRNKARAVLRAIATAHHAQETF

TrpD MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQ
SEQ ID NO: 77 TLIERLATMSNPVLMLSPGPGVPSEAGCMPELLTRLRG
KLPIIGICLGHQAIVEAYGGYVGQAGEILHGKASSIEHD
GQAMFAGLTNPLPVARYHSLVGSNIPAGLTINAHFNG
MVMAVRHDADRVCGFQFHPESILTTQGARLLEQTLA
WAQQKLEPTNTLQPILEKLYQAQTLS QQESHQLFS AV
VRGELKPEQLAAALVSMKIRGEHPNEIAGAATALLEN
AAPFPRPDYLFADIVGTGGDGSNSINISTASAFVAAAC
GLKVAKHGNRS VS S KS GS SDLLAAFGINLDMNADKSR
QALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLF
NVLGPLINPAHPPLALIGVYSPELVLPIAETLRVLGYQR
AAVVHSGGMDEVSLHAPTIVAELHDGEIKSYQLTAED
FGLTPYHQEQLAGGTPEENRDILTRLLQGKGDAAHEA
AVAANVAMLMRLHGHEDLQANAQTVLEVLRS GS AY
DRVTALAARG
TrpC MQTVLAKIVADKAIWVETRKEQQPLASFQNEVQPSTR
SEQ ID NO: 79 HFYDALQGARTAFILECKKASPSKGVIRDDFDPARIAA
IYKHYASAISVLTDEKYFQGSFDFLPIVSQIAPQPILCK
DFIIDPYQIYLARYYQADACLLMLSVLDDEQYRQLAA
VAHSLEMGVLTEVSNEEELERAIALGAKVVGINNRDL
RDLSIDLNRTRELAPKLGHNVTVISESGINTYAQVREL
SHFANGFLIGSALMAHDDLNAAVRRVLLGENKVCGL
TRGQDAKAAYDAGAIYGGLIFVATSPRCVNVEQAQE
VMAAAPLQYVGVFRNHDIADVADKAKVLSLAAVQL
HGNEDQLYIDNLREALPAHVAIWKALSVGETLPARDF
QHIDKYVFDNGQGGSGQRFDWSLLNGQSLGNVLLAG
GLGADNCVEAAQTGCAGLDFNSAVESQPGIKDARLL
AS VFQTLRAY
TrpB MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQK
SEQ ID NO: 81 DPEFQAQFNDLLKNYAGRPT ALT KCQNITAGTNTTLY
LKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAET
GAGQHGVAS ALAS ALLGLKCRIYMGAKDVERQSPNV
FRMRLMGAEVIPVHS GS ATLKDACNEALRDWS GS YE
TAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILERE
GRLPDAVIACVGGGSNAIGMFADFINETDVGLIGVEPG
GHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQI
EESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEA
LEAFKTLCLHEGIIPALESSHALAHALKMMRENPEKEQ
LLVVNLSGRGDKDIFTVHDILKARGEI
TrpA MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIID
SEQ ID NO: 83 TLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGV
TPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEF
YAECEKVGVDSVLVADVPVEESAPFRQAALRHNVAPI
FICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENR
AALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAID
AGAAGAIS GS AIVKIIEQHINEPEKMLAALKAFVQPMK
AATRS

[0397] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 9A 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 9B 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 9A 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 9B or a functional fragment thereof.
[0398] In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. 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:

71 through SEQ ID NO: 83. 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: 71 through SEQ ID NO: 83.
[0399] Table 10A depicts exemplary polypeptide sequences feedback resistant AroG and TrpE. Table 10A 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 10A. Feedback resistant AroG and TrpE and tryptophanase sequences Description Sequence AroGfbr: feedback MNYQNDDLRIKEIKELLPPVALLEKFPATENAANTVAHARKAI
resistant 2-dehydro- HKILKGNDDRLLVVIGPCSIHDPVAAKEYATRLLTLREELQDE

deo xypho sphohept LLLDINDS GLPAAGEFLDMITLQYLADLMSWGAIGARTTES Q
o nate aldo las e from VHRELAS GLSCPVGFKNGTDGTIKVAIDAINAAGAPHCFLS VT
E. coli KWGHS AIVNTS GNGDCHIILRGGKEPNYSAKHVAEVKEGLNK
AGLPAQVMIDFS HANS S KQFKKQMDVCTDVCQQIAGGEKAII
SEQ ID NO: 84 GVMVESHLVEGNQSLESGEPLAYGKSITDACIGWDDTDALLR
QLAS AVKARRG
TrpEtbr: feedback MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLEFADI
resistant DS KDDLKSLLLVDS ALRIT ALS DTVTIQALS GNGEALLTLLDN
anthranilate ALPAGVENE QS PNCRVLRFPPVS PLLDEDARLC S LS VFDAFRL
synthase LQNLLNVPKEEREAMFFGGLFSYDLVAGFENLPQLS AENS CP
component I from DFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNE
E. coli LRQQLTEAAPPLPVVS VPHMRCECNQS DEEFGGVVRLLQ KAI
RAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDN
SEQ ID NO: 85 DFTLFGASPES S LKYD AT S RQIEIYPIAGTRPRGRRADGS LDRD
LDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVA
DLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGT
LS GAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIV
IRS ALVENGIATVQAGAGVVLDS VPQSEADETRNKARAVLRA
IATAHHAQETF
SerA: 2- MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL
oxoglutarate DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT
reductase from E. NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE
coli Nissle ANAKAHRGVWNKLAAGS FE ARGKKLGIIGYGHIGT QLGILAE
SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE
SEQ ID NO: 86 NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK
HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA
QENIGLEVAGKLIKYSDNGSTLS AVNFPEVSLPLHGGRRLMHI
HENRPGVLTALNKIFAEQGVNIAAQYLQTS AQMGYVVIDIEA
DEDVAEKALQAMKAIPGTIRARLLY

SerAfbr: feedback MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL
resistant 2- DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT
oxoglutarate NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE
reductase from E. ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE
coli Nissle SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE
NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK
SEQ ID NO: 87 HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA
QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI
AEARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA
DEDVAEKALQAMKAIPGTIRARLLY
TnaA: MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE
tryptophanase from DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE
E. coli SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRS KM
VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN
SEQ ID NO: 88 FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKVM
YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE
TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT
LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA
QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPA
QALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTI
PRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFT
AKLKEV
Table 10B.
fbrAroG
atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga SEQ ID NO: 256 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa fbrTrpE atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattc SEQ ID NO: 274 gcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcg cattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccct TE 555o 5o OOOE 51E 551OTE 5115 buomuuouu 5501E1510111510551 Imo 5015 51oBEEEE buoboo5oEuom 515ou 5EE 51ou 5101E000E150001E 5o 51005501E0110E00051E 5o boomooluu 5BEEETTEEouu 51E 51E 5510505 5 5EuEouoluEE boluouuoouou11551o5uo515olloo5BEE 5510 5o 55EuEuoo OISI :ON m OHS
Bo 5150 5 5EE 5E15510 51oul 5EETTE 5Euou 5BEE 5E 5510 bolul 5 5EuEo 551E
KIJIvuoS
Eularl5p5p150005o5ouroar1555oourio5Euu 5Truo55.ro5p5o5EEEEE5oo5115ar5Eu5ar5oo5Eu5lluv5BEB5E15Tr B555Tamoo5oopouRroupTuTruo5o5oo5oTraruoi5o555m5E5oo5 BmurEuarEop5o5prup515o555ool5oarEEE5arooTraro5v5p15o 15o555155aro5proo5p5opi5EE55000parE5155o5pplo5oRrolo5 5Truar5puri5Euov5BEEE1555o5B5EE55105oTuTrE5E55.ro5o5uu 55Eopr5oB55o551Trararoo5ar5lopool5Truar5ollrE51515p5oopi oarmrooTr5o5uvuoar5o55oarE55ar5000lvi5ar5oTruo55o55555 o551oTrouRro5u5o551o5o5ar515151o5o5molvv5515515pri55o5o 5o1p5vEur5p5p5olo550005EE5Trup5omr5E5EuE5o5o555v5Tr TruuRroarool5ooTrE5E5Eoari5vo5p15E515515w5o5E5vvE5p5lo ar5loppluo5rou155.roproo5oRro5551o5oo5pruuarEEE5lluv5vp Barm5v15TrE555p5oTru5p551olvo555llruo5ar155lluvol55ario 55ovov15551o5EuRruo55o5o5o5EampB555o55o55prEuarE55 1515o5515oTro5o5uRrio5Truoo5Earoo515o55o5o5urp5p5p5pr E5o551v5155p5E55o5B5plo5o5ouTruEopp5oaro5arumr155000 Tr555o5o5uRro55o55o5v551ov51155.roTruuarE55ovi5p1B5p551 vp5o155prEEEE5m5oo5oRrov515ar5Eapalowooari5000Tr5o 5loo55ovolpr0005v5o5ooTrooTrauuumvuarav5v551o5o5o5 5uRraromraoTrarmaroup551o5m515olloo5EuE551o5o55EuRroo gsz :0N m oas uo515o55EE5E155p5p1B5EulTr5Euar5EuE5E55p5ov155ERro55Tr 100S
Elo 115ar5E55Epro5TroTro5o5oaroo5llup5o5o5pri5p5o50005EEETru 15opruaar5oo5EE55o15m5oop5lopapool5E15o551o5155oo5u.ro 515oaroo5ov155arEEE55155p5o55olo5oov5151vo5loarar5olow 5o55Tro5o5oarmvp55E155o5o55o55ario5m55o5ar5o15o155EE5 uo5Eu5oo5BEEB5m5vio5o5oul5EuE5oaro5155o5EEB5ar5555vw avi5loo5o5oarpo5aro5l0005ar5olow5aroo5o5p5E5155115115o5 000pi5Eloaro5v515omplarp5oar5B5ERroarolov5oo5o15ario5o o5m55ooararo5mro5aro55loar5vEl50005olov551551o5v5low arapplo5E5EuEvoTr5oari5o5v5E55praoTro5oo5Ear5oloar5E
5Ear55p5o1155v5oo515o15o155o5arool5ararE55oo5BE5000mpu 5E51Tamo5oo5uoaroo5ar5v15E.rop5oB5EuE55oo5o15o5o5511151 ooaroBv5TruTr55.ro5vBBB5Troul50005u000vE15E5EEEEE5p515o Eurloo5m55p5oarol50005poo5plopm5oo5olowoo5155155.roop BEEE5E55oo5o5our5o5EuRruo5B5m5o515E15155155oB5E5EE5Tr5 o5E5uoarui5vE515115o5vvo5oo515oom55155oo5p5oo5oo5o5oo 5Eu5oar5pruo5uoi5oularE5arE5loo5olo5prolo15aruarEEEE5EE5 EE5Tru5oolo5B151oo5uoo55.rolvi5opro5EEEEEEE5uovoar5BE515 5v5p5oRrap5oloviBB5lopv5poo5p5ETrEEE55o5uol5pruo5o armrEEE5mr555o5515Boar5vpolop5po55o55opop5Truo5EE5E
5arE5EE55Eu5oari5vE5B5loTrE5Earivip5oopp5ar5B1B55oppo op5vpo50005ar5Eav551o5prool5m151oo5000lp5o5pri5o5oo 51ouRroaroTruarE5vEuE55151555o5loo5Boo5arEv55priarar5B5 099I0/LIOZSI1IIDd cccggtatttaacgcaccgttctcaaatacgcgctctgttgcgg agctggtg attggcg aa ctgctgctgctattgcgcggcgtgccag aagccaatgctaaagcgcatcgtggcgtgtgg aacaaactggcggcgggttcttttg aagcgcgcggcaaaaagctgggtatcatcggcta cggtcatattggtacgcaattgggcattctggctg aatcgctggg aatgtatgtttacttttatg atattg aaaacaaactgccgctgggcaacgccactcaggtacagcatctttctg acctgc tg aatatg agcg atgtggtg agtctgcatgtaccag ag aatccgtccaccaaaaatatg a tgggcgcg aaag ag atttcgctaatg aagcccggctcgctgctg attaatgcttcgcgcg gtactgtggtgg atattccagcgctgtgtg acgcgctggcg agcaaacatctggcgggg gcggcaatcg acgtattcccg acgg aaccggcg accaatagcg atccatttacctctcc gctgtgtg aattcg acaatgtccttctg acgccacacattggcggttcg actcagg aagcg cagg ag aatatcggcttgg aagttgcgggtaaattg atcaagtattctg acaatggctcaa cgctctctgcggtg aacttcccgg aagtctcgctgccactgcacggtgggcgtcgtctg at g cacatcGCTg aaGCTcg tccgggcg tgctaactgcgctcaacaaaatttttgccg a gcagggcg tcaacatcgccgcgcaatatctacaaacttccgcccag atggg ttatg tag t tattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatgaaagct attccgggtaccattcgcgcccgtctgctgtactaa [0400] In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 10B.
In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 10B. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 10B. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 10B. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 96%, 97%, 98%, or 99%
identity with one or more sequences of Table 10B. 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 10B. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 10B.
[0401] In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ
ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 256. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:
256.
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 SEQ ID NO: 256.
In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ
ID NO: 256. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 256.
[0402] In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. 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:
84 through SEQ ID NO: 87. 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: 84 through SEQ ID NO: 87.
[0403] In some embodiments, the endogenous TnaA polypeptide comprising SEQ
ID NO: 88 is mutated or deleted.

[0404] To improve acetate production, while maintaining high levels of tryptophan production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Deletions which may be introduced therefore include deletion of adhE, ldh, and frd.
Thus, in certain embodiments, the genetically engineered bacteria comprise one or more tryptophan production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0405] In some embodiments, the genetically engineered bacteria comprise one or more tryptophan production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA
gene and the adhE gene.
[0406] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA and rdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
[0407] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0408] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0409] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions.
[0410] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of tryptophan production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for tryptophan production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for tryptophan synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE
genes.
[0411] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA
gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta, ldhA
and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
[0412] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in the endogenous pta and ldhA genes.
[0413] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in the endogenous pta and frdA genes.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and AtrpR, AtnaA, and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0414] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0415] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions.
[0416] 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 and others described herein. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0417] n some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.
Producing Kynurenic Acid [0418] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD
patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene or genes for producing kynurenic acid. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (also referred to as kynurenine aminotransferases (e.g., KAT I, II, III)).
[0419] In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter.
In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0420] 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.
[0421] 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.
[0422] 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.
[0423] In one embodiment, 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 in the figures 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 IDO1 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 ID01. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with TD02. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine--oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of idol and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with idol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2.
[0424] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from homo sapiens.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 (Kynurenine--oxoglutarate transaminase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 from homo sapiens). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine--oxoglutarate transaminase 3) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from homo sapiens.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cclbl and/or cc1b2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of idol and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of. cclbl and/or cc1b2 and/or aadat and/or got2.
[0425] 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 the figures and the examples 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.
[0426] In some embodiments, the one or more genes for producing kynurenic acid are modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell.
[0427] To improve acetate production, while maintaining high levels of kynurenic acid production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA
to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more kynurenic acid production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0428] In some embodiments, the genetically engineered bacteria comprise one or more kynurenic acid production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA
gene and the adhE gene.
[0429] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous adhE
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous frdA
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA
and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA
genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA
gene, the frdA gene and the adhE genes.
[0430] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0431] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions.
[0432] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of kynurenic acid production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for kynurenic acid production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for kynurenic acid synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes.
[0433] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA
gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta and adhE genes.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta, frdA
and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
[0434] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0435] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions.
[0436] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0437] . In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0438] 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 and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome.
Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.

Producing Indole Tryptophan Metabolites and Tryptamine [0439] 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 the figures.
[0440] In some embodiments, the genetically engineered bacteria comprise genetic circuitry for converting tryptophan to tryptamine. In some embodiments, the engineered bacteria comprise gene sequence encoding Tryptophan decarboxylase, e.g., from Catharanthus roseus. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetaldehyde and FICZ from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S.
cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli, taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), sta0 (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA
(Monoamine oxidase, e.g., from E. coli). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetonitrile from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynurenine from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following:
ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynureninic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: ID01(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid:
Kynurenine formamidase, e.g., from mouse), 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 CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tnaA (tryptophanase, e.g., from E. coli). In some embodiments, the engineered bacteria comprise genetic circuitry for producing 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 gene sequence encoding pne2 (myrosinase, e.g., from Arabidopsis thaliana).
In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli, taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), sta0 (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC
(Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae), iadl ( Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AA01 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g.,from Catharanthus roseus and/or Clostridium sporogenes), tynA
(Monoamine oxidase, e.g., from E. coli), iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AA01 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E.
coli, taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), sta0 (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 ( indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IaaM
(Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi), iaaH
(Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana, cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana), nitl (Nitrilase, e.g., from Arabidopsis thaliana), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprises trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC
(Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-acetaldehyde into indole-3-acetat [0441] In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. In some embodiments, the engineered bacteria produces tryptamine.
Tryptophan is optionally produced from chorismate precursor, and the bacteria optionally comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B
and/or FIG.
40C and/or FIG. 40D. Additionally, the bacteria comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.
[0442] In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-acetate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG.
40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. 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 (indo1-3y1)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.
[0443] In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-propionate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D.
Additionally, the strain comprises a circuit as described in FIG. 48, comprising trpDH
(Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indo1-3y1)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 indo1-3-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 AcuI: (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 comprisefldH/ and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indo1-3-yl)pyruvate into indole-3-lactate).
[0444] In some embodiments, the engineered bacteria comprises genetic circuitry for the production of indole-3-propionic acid (IPA). In some embodiments, the engineered bacteria comprises gene sequence encoding 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. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0445] In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-propionic acid (IPA), indole acetic acid (IAA), and/or tryptamine synthesis(TrA) circuits. In some embodiments, the engineered bacteria comprise gene sequence encoding one or more of the following: TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldHl/F1dH2: 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; AcuI: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. 1pdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae;
ladl: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc:
Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.
[0446] In some embodiments, the engineered bacteria comprise genetic circuitry for producing (indo1-3-yl)pyruvate (IPyA). In some embodiments, the engineered bacteria comprise gene sequence encoing one or more of the following: tryptophan dehydrogenase (EC 1.4.1.19) (enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indo1-3-yl)pyruvate (IPyA), NH3, NAD(P)H and H ));
Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g., Clostridium sporogenes or Lactobacillus casei) (converts (indo1-3y1)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+); Indole-3-propionyl-CoA:indole-3-lactate CoA
transferase (F1dA ) (converts indole-3-lactate (ILA) and indo1-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA); Indole-3-acrylyl-CoA reductase (F1dD ) and acrylyl-CoA reductase (AcuI) (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); Indole-3-pyruvate decarboxylase (1pdC:) (converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAA1d)); ladl: Indole-3-acetaldehyde dehydrogenase (coverts Indole-3-acetaldehyde (IAA1d) into Indole-3-acetic acid (IAA)); Tdc:
Tryptophan decarboxylase (converts tryptophan (Trp) into tryptamine (TrA)). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria.
In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0447] In any of the described embodiments, any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter.
In certain embodiments, the one or more cassettes are under the control of constitutive promoters. 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 (A thyA; thymidine dependence).
[0448] In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG.
40 (A-D) and FIG. 44 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA
gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0449] 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.
Exemplary inducible promoters which may control the expression of the biosynthetic cassettes include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS
promoters), and promoters induced by a metabolite characteristic of a disorder described herein, or that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0450] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following:
(1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.
Tryptamine [0451] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which 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 (IaaM) and indole-3-acetamide hydrolase (IaaH), which constitute the indole-3-acetamide (IAM) pathway, as described in the figures and examples.

[0452] A non-limiting example of such as strain is shown in FIG. 41A. Another non-limiting example of such as strain is shown in FIG. 43A. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Clostridium sporgenenes. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from Ruminococcus Gnavus.
[0453] Table 15, Table 11A, and Table 12 lists exemplary sequences for tryptamine production in genetically engineered bacteria.
[0454] 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. 40, FIG. 44A and/or FIG. 44B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 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.
[0455] To improve acetate production, while maintaining high levels of tryptamine production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
Deletions which may be introduced therefore include deletion of adhE, ldh, and frd.
Thus, in certain embodiments, the genetically engineered bacteria comprise one or more tryptamine production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0456] In some embodiments, the genetically engineered bacteria comprise one or more tryptamine production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA
gene and the adhE gene.
[0457] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA and rdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
[0458] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA
gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE
genes.
[0459] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0460] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions.
[0461] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of tryptamine production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for tryptamine production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for tryptamine synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE
genes.
[0462] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA
gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta and adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta and ldhA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta, ldhA
and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.

[0463] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation in the endogenous pta and ldhA genes.
[0464] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation in the endogenous pta and frdA genes.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and AtrpR and AtnaA, and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0465] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0466] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions.
[0467] In some embodiments, the genetically engineered bacteria are capable of producing 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 and others described herein.
Indole-3-acetaldehyde and FICZ
[0468] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 41B.
[0469] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or sta0 or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or sta0 or trpDH and ipdC.

[0470] Further exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 41C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA.
[0471] 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. 40, FIG. 44A and/or FIG. 44B
and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 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.
[0472] To improve acetate production, while maintaining high levels of Indole-acetaldehyde and/or FICZ production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production).
Non-limiting examples of competing such competing metabolic arms are frdA
(converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more Indole-3-acetaldehyde and/or FICZ production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0473] In some embodiments, the genetically engineered bacteria comprise one or more Indole-3-acetaldehyde and/or FICZ production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene.
[0474] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE
genes.
[0475] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0476] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions.

[0477] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of Indole-3-acetaldehyde and/or FICZ production.
Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for Indole-3-acetaldehyde and/or FICZ
production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for Indole-3-acetaldehyde and/or FICZ synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE
genes.
[0478] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ
and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ
and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta, frdA
and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
[0479] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0480] In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions.
[0481] In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde 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.
[0482] In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
Indole-3-acetic acid [0483] 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 the tables. 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.
[0484] The genetically engineered bacteria may comprise any suitable gene for producing Indole-3-aldehyde and/or Indole Acetic Acid and/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 Acid and/or Tryptamine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite, e.g., comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acid and/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.
[0485] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetic acid.
[0486] Non-limiting example of such gene sequence(s) are shown in FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E, and FIG. 43B and FIG. 43E.
[0487] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from S. cerevisae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 from streptomyces sp. TP-A0274). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108).
In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AA01 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AA01 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taal and/or sta0 and/or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC
(Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from iadl and/or aaol.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC
and/or taal and/or sta0 and in combination with one or more sequences encoding enzymes selected from iadl and/or aaol (see, e.g., FIG. 42A).
[0488] Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 42B. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iadl from Ustilago maydis). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AA01 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AA01 from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iadl and/or aaol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iadl and/or aaol. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iadl and/or aaol.
[0489] Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 42C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole-3-pyruvate monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from Enterobacter cloacae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2.
In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taal and yuc2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode sta0 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and yuc2.. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC
or taal or sta0 or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taal or sta0 or trpDH and yuc2.
[0490] Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 42D. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH
from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH.
[0491] Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 42E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis thaliana.
In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nitl (Nitrilase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nitl from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi),In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13, and nitl and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nitl and iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13 and nitl and iaaH.

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Claims (37)

1. A bacterium comprising at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacterium comprises an endogenous pta gene which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.

2. The bacterium of claim 1, wherein the bacterium comprises an endogenous adhE
gene which is knocked down via mutation or deletion.

3. The bacterium of claim 1 or claim 2, wherein the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion.

4. The bacterium of any of claims 1-3, wherein the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.

5. The bacterium of any of claims 1-4, wherein the at least one gene cassette comprises ter, thiAl, hbd, crt2, pbt, and buk genes.

6. The bacterium of any of claims 1-4, wherein the at least one gene cassette comprises ter, thiAl, hbd, crt2, and tesb genes.

7. A bacterium comprising a biosynthetic pathway for producing acetate, wherein the bacterium comprises an endogenous adhE gene which is knocked down via mutation or deletion.

8. A bacterium comprising a biosynthetic pathway for producing acetate, wherein the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion.

9. A bacterium comprising a biosynthetic pathway for producing acetate, wherein the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.

10. The bacterium of claim 7 or claim 9, wherein the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion.

11. The bacterium of claim 7 or claim 8, wherein the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.

12. The bacterium of claim 7, wherein the bacterium comprises an endogenous ldhA
gene and an endogenous frd gene, both of which genes are knocked down via mutation and/or deletion.

13. The bacterium of any one of claims 7-12, wherein the biosynthetic pathway for producing acetate is a native biosynthetic pathway endogenous to the bacterium.

14. The bacterium of any one of claims 7-12, wherein the biosynthetic pathway for producing acetate is a non-native biosynthetic pathway.

15. The bacterium of claim 14, wherein the bacterium comprises at least one gene or gene cassette encoding the non-native biosynthetic pathway for producing acetate, wherein the at least one gene or gene cassette for producing acetate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature

16. The bacterium of any one of claims 1-6 and claim 15, wherein the promoter is induced by exogenous environmental conditions found in a mammalian gut.

17. The bacterium of claim 16, wherein the promoter is induced under low-oxygen or anaerobic conditions.

18. The bacterium of claim 17, wherein the promoter is a FNR-responsive promoter, an ANR-responsive promoter, or a DNR-responsive promoter.

19. The bacterium of claim 18, wherein the promoter is a FNR-responsive promoter.

20. The bacterium of any one of claims 1-6 and claim 15, wherein the promoter is induced by the presence of reactive nitrogen species.

21. The bacterium of claim 20, wherein the promoter is a NsrR-responsive promoter, NorR-responsive promoter, or a DNR-responsive promoter.

22. The bacterium of any one of claims 1-6 and claim 15, wherein the promoter is induced by the presence of reactive oxygen species.

23. The bacterium of claim 22, wherein the promoter is a OxyR-responsive promoter, PerR-responsive promoter, OhrR-responsive promoter, SoxR-responsive promoter, or a RosR-responsive promoter.

24. The bacterium of any one of claims 1-6 or claims 14-15, wherein the gene and/or gene cassette is located on a chromosome in the bacterium.

25. The bacterium of any one of claims 1-6 or claims 14-15, wherein the at least one gene and/or gene cassette is located on a plasmid in the bacterium.

26. The bacterium of any one of claims 1-25, wherein the bacterium is a probiotic bacterium.

27. The bacterium of claim 26, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.

28. The bacterium of claim 27, wherein the bacterium is Escherichia coli strain Nissle.

29. The bacterium of any one of claims 1-28, wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.

30. The bacterium of claim 29, wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.

31. A pharmaceutically acceptable composition comprising the bacterium of any one of claims 1-30; and a pharmaceutically acceptable carrier.

32. The composition of claim 31 formulated for oral or rectal administration.

33. A method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, the composition of any one of claims 31 or 32.

34. A method of treating a disease or condition associated with gut inflammation and/or compromised gut barrier function comprising the step of administering to a patient in need thereof, the composition of any one of claims 31 or 32.

35. The method of claim 33, wherein the autoimmune disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, 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 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, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds 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.

36. The method of claim 35, wherein the autoimmune disorder is selected from the group consisting of type 1 diabetes, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, celiac disease, and ankylosing spondylitis.

37. The method of claim 34, wherein the disease or condition is selected from an inflammatory bowel disease, including Crohn's disease and ulcerative colitis, and a diarrheal disease.