Bacteria Engineered to Treat Diseases that Benefit from Reduced Gut Inflammation and/or Tighten Gut Mucosal Barrier

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

[0001] This application is a continuation of U.S. application Ser. No. 15/868,487, filed Jan. 11, 2018, which is a continuation of U.S. application Ser. No. 14/998,376, filed Dec. 22, 2015, which claims benefit of priority to U.S. Provisional No. 62/095,415, filed Dec. 22, 2014; U.S. Provisional No. 62/127,131, filed Mar. 2, 2015; U.S. Provisional No. 62/248,825, filed Oct. 30, 2015; U.S. Provisional No. 62/256,044, filed Nov. 16, 2015, which are incorporated herein by reference in their entirety.

[0002] 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 reduce inflammation in the gut and/or enhance gut barrier function, particularly in the presence of reactive nitrogen species. In some embodiments, the genetically engineered bacteria reduce gut inflammation and/or enhance 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.

[0003] 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.RTM. (Cohen et al., 2014). Drawbacks from this approach are associated with systemic immunosuppression, which includes greater susceptibility to infectious disease and cancer.

[0004] Other approaches have focused on treating compromised barrier function by supplying the short-chain fatty acid butyrate via enemas. Recently, several groups demonstrated the importance of short-chain fatty acid production by commensal bacteria in regulating the immune system in the gut (Smith et al., 2013). They showed 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.

[0005] Compromised gut barrier function also plays "a central part . . . in autoimmune diseases pathogenesis" (Lerner et al., 2015a; Lerner et al., 2015b; Fasano et al., 2005; Fasano et al., 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) 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 . . . as drivers of the autoimmune cascade" (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).

[0006] 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). 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 gastrointestinal tract are potential therapeutic mechanisms for the treatment or prevention of autoimmune disorders.

[0007] 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. While these engineered microbes have shown efficacy in some pre-clinical models, efficacy in patients has not been observed. One main reason why these engineered microbes have not been successful in treating patients is that their viability and stability are compromised, because they constitutively produce large amounts of non-native proteins, e.g., human interleukin. Thus, there remains a great need for additional therapies that reduce gut inflammation, enhance gut barrier function, and/or treat autoimmune disorders, and that avoid undesirable side effects.

[0008] Reactive nitrogen species (RNS) such as nitric oxide are produced at sites of inflammation and are intimately associated with the disease process. Certain bacterial transcription factors have evolved to sense RNS and regulate the expression of a number of proteins that protect the bacterial DNA from their damaging effects.

[0009] The genetically engineered bacteria of the invention are capable of producing therapeutic anti-inflammation and/or gut barrier enhancer molecules, particularly in the presence of RNS. The genetically engineered bacteria are functionally silent until they reach an environment containing local RNS, wherein expression of the therapeutic molecule is induced. In certain embodiments, the genetically engineered bacteria are 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.

[0010] The genetically engineered bacteria of the invention produce a therapeutic molecule under the control of a RNS-responsive regulatory region and a corresponding RNS-sensing transcription factor. In some embodiments, the therapeutic molecule is butyrate; in environment containing local RNS, 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. The genetically engineered bacteria of the invention produce their therapeutic effect only in environments environment containing local ROS, thereby lowering the safety issues associated with systemic exposure.

BRIEF DESCRIPTION OF THE FIGURES

[0011] FIGS. 1A and 1B depict the construction and gene organization of two exemplary plasmids each comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrate operon. FIG. 1A shows the pLogic031-nsrR-norB-butyrate operon construct, and FIG. 1B shows the pLogic046-nsrR-norB-butyrate operon construct.

[0012] FIG. 2 depicts the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In the upper panel, in the absence of NO, the NsrR transcription factor (gray circle, "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In the lower panel, 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 gray arrows and black squiggles) and ultimately to the production of butyrate.

[0013] FIG. 3 depicts the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In the upper panel, in the absence of NO, the NsrR transcription factor (gray circle, "NsrR") binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In the lower panel, 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 gray arrows and black squiggles) and ultimately to the production of butyrate.

[0014] FIG. 4 depicts the nucleic acid sequence of an exemplary RNS-regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrate operon (pLogic031-nsrR-norB-butyrate operon construct; SEQ ID NO: 1). The sequence encoding NsrR is underlined and bolded, and the NsrR binding site, i.e., a regulatory region of norB is .

[0015] FIG. 5 depicts the nucleic acid sequence of an exemplary RNS-regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrate operon (pLogic046-nsrR-norB-butyrate operon construct; SEQ ID NO: 2). The sequence encoding NsrR is underlined and bolded, and the NsrR binding site, i.e., a regulatory region of norB is .

[0016] FIG. 6 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct comprising a tet promoter and butyrate operon (pLogic031-tet-butyrate operon construct; SEQ ID NO: 12). The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are .

[0017] FIG. 7 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct comprising a tet promoter and butyrate operon (pLogic046-tet-butyrate operon construct; SEQ ID NO: 13). The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are .

[0018] FIG. 8 depicts synthetic biology circuits comprising parallel pathways, in vivo activation switches, auxotrophy and kill switches, and peptide/protein export.

[0019] FIG. 9 depicts synthetic biology safety designs, e.g., auxotrophy and kill switches.

[0020] FIG. 10 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome.

[0021] FIG. 11 depicts a schematic for inflammatory bowel disease (IBD) therapies that target pro-inflammatory neutrophils and macrophages and regulatory T cells (Treg), restore epithelial barrier integrity, and maintain mucosal barrier function.

[0022] FIGS. 12 A, B, and C depict ATC or nitric oxide-inducible reporter constructs. 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 (FIGS. 12A and 12C) or the nitric oxide inducible PnsrR-GFP reporter construct (FIGS. 12B and 12C) induced across a range of concentrations. Promoter activity is expressed as relative florescence units.

[0023] FIG. 13 the sequence of an exemplary nitric oxide-inducible reporter construct. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are .

[0024] FIG. 14 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. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice.

[0025] FIG. 15A depicts a schematic diagram of a wild-type clbA construct (SEQ ID NO: 15). FIG. 15B depicts a schematic diagram of a clbA knockout construct (SEQ ID NO: 16).

[0026] FIG. 16 depicts a map of exemplary integration sites within the E. coli 1917 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.

DESCRIPTION OF EMBODIMENTS

[0027] The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of reducing gut inflammation, enhancing gut barrier function, and/or and treating or preventing autoimmune disorders. The genetically engineered bacteria of the invention comprise a gene encoding a non-native anti-inflammation and/or gut barrier function enhancer molecule, or a gene cassette encoding a biosynthetic pathway for producing an anti-inflammation and/or gut barrier function enhancer molecule. The gene or gene cassette is further linked to a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species.

[0028] 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.

[0029] 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 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. "Diarrheal diseases" include, but are not limited to, acute watery diarrhea, e.g., cholera, acute bloody diarrhea, e.g., dysentery, and persistent diarrhea. Related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis.

[0030] 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.

[0031] 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, 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 (UP), 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.

[0032] "Anti-inflammation molecules" and/or "gut barrier function enhancer molecules" include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, GLP-2, IL-10, IL-27, TGF-.beta.1, TGF-.beta.2, N-acylphosphatidylethanolamines (NAPES), elafin (also called peptidase inhibitor 3 and SKALP), and trefoil factor. 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-.alpha., IFN-.gamma., IL-1.beta., IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2. A molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2. A molecule may be both anti-inflammatory and gut barrier function enhancing. An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene. Alternatively, an anti-inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., butyrate. These molecules may also be referred to as therapeutic molecules.

[0033] 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-inflammation and/or gut barrier function enhancer molecule, e.g., butyrate. 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.

[0034] 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, thiA1, 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. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiA1 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 thiA1, 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.

[0035] 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, etfA, 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). In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrA.sup.fbr, thrB, thrC, ilvA.sup.fbr, aceE, aceF, and lpd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltransferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase. 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 butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.

[0036] 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 CO.sub.2+H.sub.2 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.

[0037] "Reactive nitrogen species" and "RNS" are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO.cndot.), peroxynitrite or peroxynitrite anion (ONOO.sup.-), nitrogen dioxide (.cndot.NO.sub.2), dinitrogen trioxide (N.sub.2O.sub.3), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOCO.sub.2.sup.-) (unpaired electrons denoted by .cndot.).

[0038] Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics. "RNS-inducible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. Thus, RNS induces expression of the gene or gene cassette.

[0039] "RNS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene or gene cassette. Thus, RNS derepresses expression of the gene or gene cassette.

[0040] "RNS-repressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene or gene cassette. Thus, RNS represses expression of the gene or gene cassette.

[0041] A "RNS-responsive regulatory region" refers to a RNS-inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 1.

[0042] 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, and the tunable regulatory region may be a RNS-responsive regulatory region. The tunable regulatory region may be operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. For example, 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.

[0043] A gene or gene cassette for producing a therapeutic molecule 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. "Directly controlled" refers to a RNS-inducible or RNS-derepressible regulatory region, in which the regulatory region is operatively linked to said gene or gene cassette; in the presence of RNS, the therapeutic molecule is expressed. "Indirectly controlled" refers to a RNS-repressible regulatory region, wherein a RNS-sensing repressor inhibits transcription of a second repressor, which inhibits the transcription of the gene or gene cassette for producing a therapeutic molecule; in the presence of RNS, the second repressor does not inhibit transcription of said gene or gene cassette, and the therapeutic molecule is expressed. "Operatively linked" refers a nucleic acid sequence, e.g., a gene or gene cassette for an anti-inflammation and/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.

TABLE-US-00001 TABLE 1 Examples of RNS-sensing transcription factors and RNS-responsive genes RNS-sensing Primarily Examples of responsive transcription capable genes, promoters, and/or factor: of sensing: regulatory regions: NsrR NO norB, aniA, nsrR, hmpA, ytfE, ygbA, hcp, hcr, nrfA, aox NorR NO norVW, norR DNR NO norCB, nir, nor, nos

[0044] As used herein, a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence, e.g., a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et 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. The non-native nucleic acid sequence may be present on a plasmid or chromosome.

[0045] "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.

[0046] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, 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. Pat. Nos. 6,835,376; 6,203,797; 5,589,168; 7,731,976).

[0047] "Probiotic" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Pat. Nos. 5,589,168; 6,203,797; 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.

[0048] 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 butyrogenic gene cassette, that 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 modified bacterium comprising a butyrogenic gene cassette, in which the plasmid or chromosome carrying the butyrogenic gene cassette is stably maintained in the host cell, such that butyrate can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro and/or in vivo.

[0049] As used herein, the term "treat" and its cognates refer to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, "treat" refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "treat" refers to inhibiting the progression of a disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, "treat" refers to slowing the progression or reversing the progression of a disease or disorder. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease or disorder.

[0050] 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 or disorder. In some instances, the "initial colonization of the newborn intestine is particularly relevant to the proper development of the host's immune and metabolic functions and to determine disease risk in early and later life" (Sanz et al., 2015). In some embodiments, early intervention (e.g., prenatal, perinatal, neonatal) using the genetically engineered bacteria of the invention may be sufficient to prevent or delay the onset of the disease or disorder.

[0051] As used herein a "pharmaceutical composition" refers to a preparation of genetically engineered bacteria of the invention with other components such as a physiologically suitable carrier and/or excipient.

[0052] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.

[0053] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[0054] 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. 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 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.

[0055] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0056] 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.

[0057] Bacteria

[0058] The genetically engineered bacteria of the invention comprise a gene encoding a non-native anti-inflammation and/or gut barrier function enhancer molecule, or a gene cassette encoding a non-native biosynthetic pathway capable of producing an anti-inflammation and/or gut barrier function enhancer molecule, wherein the gene or gene cassette is 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 reactive nitrogen species. In some embodiments, the gene or gene cassette is an additional copy of a native gene or gene cassette. In some embodiments, the gene or gene cassette is from a different species. In some embodiments, the gene or gene cassette is operably linked to a directly or indirectly inducible promoter. In some embodiments, the inducible promoter is not associated with the gene or gene cassette in nature.

[0059] In some embodiments, the genetically engineered bacteria are naturally non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.

[0060] In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive 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" (Reinter et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle "lacks prominent virulence factors (e.g., E. coli .alpha.-hemolysin, P-fimbrial adhesins)" (Schultz, 2008), and E. coli Nissle "does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, 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).

[0061] 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 from Peptoclostridium difficile have been expressed in Escherichia coli (Aboulnaga et al., 2013).

[0062] Reducing Gut Inflammation, Tightening Gut Mucosal Barrier, and/or Treating or Preventing Autoimmune Disorders

[0063] The genetically engineered bacteria of the invention comprise a gene encoding a non-native anti-inflammation and/or gut barrier function enhancer molecule, or a gene cassette encoding a biosynthetic pathway capable of producing an anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the molecule is selected from the group consisting of a short-chain fatty acid, butyrate, propionate, acetate, GLP-2, IL-10, IL-27, TGF-.beta.1, TGF-.beta.2, elafin (also known as peptidase inhibitor 3 or SKALP), and trefoil factor. 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.

[0064] In some embodiments, the genetically engineered bacteria of the invention express an anti-inflammation and/or gut barrier function enhancer molecule 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 an anti-inflammation and/or gut barrier function enhancer molecule, e.g., butyrate, that is synthesized by a biosynthetic pathway requiring multiple genes.

[0065] In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and produce butyrate in the presence of RNS. Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. The genetically engineered bacteria may include any suitable set of butyrogenic genes. 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, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013), and produce butyrate in the presence of RNS. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, 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 produce butyrate in the presence of RNS. For example, in some embodiments, the genetically engineered bacteria comprise bcd2, etfB3, etfA3, and thiA1 from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.

[0066] The gene products of the bcd2, etfA3, and etfB3 genes in Clostridium difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant. 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, etfB3, and etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the genetically engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in the presence of RNS (see, e.g., FIG. 5). 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, the genetically engineered bacteria of the invention comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile; ter, e.g., from Treponema denticola; one or more of bcd2, etfB3, and etfA3, e.g., from Peptoclostridium difficile; and produce butyrate in the presence of RNS. 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 the presence of RNS. 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.

[0067] In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and produce propionate in the presence of RNS. 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 may include any suitable set of propionate biosynthesis genes. 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 propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway propionate biosynthesis genes, e.g., thrA.sup.fbr, thrB, thrC, ilvA.sup.fbr, aceE, aceF, and lpd, and optionally further comprise tesB.

[0068] 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 biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a synthetic 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. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. In some embodiments, the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and produce propionate in low-oxygen conditions. 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 low-oxygen conditions.

[0069] In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and produce acetate in the presence of RNS. 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 et al., 2008). The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. 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 Thermoacetogenium. 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 produce acetate in the presence of RNS. 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 the presence of RNS.

[0070] In some embodiments, the genetically engineered bacteria of the invention express IL-10 in the presence of RNS. Interleukin-10 (IL-10) is a class 2 cytokine, a category which includes cytokines, interferons, and interferon-like molecules, such as IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, IL-29, IFN-.alpha., IFN-.beta., IFN-6, IFN-.epsilon., IFN-.kappa., IFN-.tau., IFN-.omega., and limitin. IL-10 is an anti-inflammatory cytokine that signals through two receptors, IL-10R1 and IL-10R2. Deficiencies in IL-10 and/or its receptors are associated with IBD and intestinal sensitivity (Nielsen 2014). Bacteria expressing IL-10 or protease inhibitors may ameliorate conditions such as Crohn's disease and ulcerative colitis (Simpson et al., 2014). The genetically engineered bacteria may comprise any suitable gene encoding IL-10, e.g., human IL-10. In some embodiments, the gene encoding IL-10 is modified and/or mutated, e.g., to enhance stability, increase IL-10 production, and/or increase anti-inflammatory potency in the presence of RNS.

[0071] In some embodiments, the genetically engineered bacteria of the invention express GLP-2 or proglucagon in the presence of RNS. Glucagon-like peptide 2 (GLP-2) is produced by intestinal endocrine cells and stimulates intestinal growth and enhances gut barrier function. Post-translational proteolytic cleavage of proglucagon produces GLP-2 and GLP-1. GLP-2 administration has therapeutic potential in treating IBD, short bowel syndrome, and small bowel enteritis (Yazbeck et al., 2009). The genetically engineered bacteria may comprise any suitable gene encoding GLP-2 or proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-2 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et al., 2009). In some embodiments, the gene encoding GLP-2 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-2 production, and/or increase gut barrier enhancing potency in the presence of RNS.

[0072] In some embodiments, the genetically engineered bacteria of the invention express a molecule that is capable of inhibiting a pro-inflammatory molecule. The genetically engineered bacteria may express any suitable inhibitory molecule, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA, that is capable of neutralizing one or more pro-inflammatory molecules, e.g., TNF, IFN-.gamma., IL-1.beta., IL-6, IL-8, IL-17, or chemokines (Keates et al., 2008; Ahmad et al., 2012). The genetically engineered bacteria may inhibit one or more pro-inflammatory molecules, e.g., TNF, IL-17.

[0073] RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in plants and animals. RNAi is activated when microRNA (miRNA), double-stranded RNA (dsRNA), or short hairpin RNA (shRNA) is processed into short interfering RNA (siRNA) duplexes (Keates et al., 2008). RNAi can be "activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver shRNA to target cells" such as mammalian cells (Keates et al., 2008). In some embodiments, the genetically engineered bacteria of the invention induce RNAi-mediated gene silencing of one or more pro-inflammatory molecules in the presence of RNS. In some embodiments, the genetically engineered bacteria produce siRNA targeting TNF in the presence of RNS.

[0074] Single-chain variable fragments (scFv) are "widely used antibody fragments . . . produced in prokaryotes" (Frenzel et al., 2013). scFv lacks the constant domain of a traditional antibody and expresses the antigen-binding domain as a single peptide. Bacteria such as Escherichia coli are capable of producing scFv that target pro-inflammatory cytokines, e.g., TNF (Hristodorov et al., 2014). In some embodiments, the genetically engineered bacteria of the invention express a binding protein for neutralizing one or more pro-inflammatory molecules in the presence of RNS. In some embodiments, the genetically engineered bacteria produce scFv targeting TNF in the presence of RNS. In some embodiments, the genetically engineered bacteria produce both scFv and siRNA targeting one or more pro-inflammatory molecules in the presence of RNS (see, e.g., Xiao et al., 2014).

[0075] One of skill in the art would appreciate that additional genes and gene cassettes capable of producing anti-inflammation and/or gut barrier function enhancer molecules are known in the art and may be expressed by the genetically engineered bacteria of the invention. In some embodiments, the gene or gene cassette for producing a therapeutic molecule also comprises additional transcription and translation elements, e.g., a ribosome binding site, to enhance expression of the therapeutic molecule.

[0076] In some embodiments, the genetically engineered bacteria produce two or more anti-inflammation and/or gut barrier function enhancer molecules. In certain embodiments, the two or more molecules behave synergistically to reduce gut inflammation and/or enhance gut barrier function. In some embodiments, the genetically engineered bacteria express at least one anti-inflammation molecule and at least one gut barrier function enhancer molecule. In certain embodiments, the genetically engineered bacteria express IL-10 and GLP-2. In alternate embodiments, the genetically engineered bacteria express IL-10 and butyrate.

[0077] RNS Tunable Regulatory Region

[0078] The genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of an anti-inflammation and/or gut barrier function enhancer molecule, thus controlling expression of the molecule relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the molecule is butyrate; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the butyrate operon, thereby producing butyrate, which exerts anti-inflammatory and/or gut barrier enhancing effects. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and butyrate production is decreased or eliminated.

[0079] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0080] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR "is an NO-responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide" (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked butyrogenic gene cassette and producing butyrate.

[0081] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) "promotes the expression of the nir, the nor and the nos genes" in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked butyrogenic gene cassette and producing butyrate. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

[0082] In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0083] In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is "an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism" (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked butyrogenic gene cassette and producing butyrate.

[0084] In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[0085] In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0086] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express an anti-inflammation and/or gut barrier function enhancer molecule. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, C1, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or gene cassette, e.g., a butyrogenic gene cassette, is expressed.

[0087] A RNS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012).

[0088] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GInRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0089] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0090] In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity.

[0091] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

[0092] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g., the NsrR gene, and a corresponding regulatory region, e.g., a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the anti-inflammatory and/or gut barrier enhancer molecule in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS-responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the anti-inflammatory and/or gut barrier enhancer molecule in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the anti-inflammatory and/or gut barrier enhancer molecule in the presence of RNS. Nucleic acid sequences of exemplary RNS-regulated constructs comprising a gene encoding NsrR and a norB promoter are shown in FIGS. 4 and 5.

[0093] The genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene(s) or gene cassette(s) capable of producing an anti-inflammation and/or gut barrier function enhancer molecule, such that said gene(s) or gene cassette(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.

[0094] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) or gene cassette(s) capable of producing an anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the gene(s) or gene cassette(s) capable of producing an anti-inflammation and/or gut barrier function enhancer molecule is present on a plasmid and operatively linked to a RNS-responsive regulatory region. In some embodiments, the gene(s) or gene cassette(s) capable of producing an anti-inflammation and/or gut barrier function enhancer molecule is present in a chromosome and operatively linked to a RNS-responsive regulatory region.

[0095] In some embodiments, any of the gene(s) or gene cassette(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of the butryogenic gene cassette may be integrated into the bacterial chromosome. Having multiple copies of the butryogenic gene cassette integrated into the chromosome allows for greater production of the butyrate and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the kill-switch circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

[0096] In some embodiments, the genetically engineered bacteria of the invention produce at least one anti-inflammation and/or gut barrier enhancer molecule in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold as compared to unmodified bacteria of the same subtype under the same conditions.

[0097] Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g., by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen).

[0098] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of an anti-inflammation and/or gut barrier enhancer molecule in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the anti-inflammation and/or gut barrier enhancer molecule. In embodiments using genetically modified forms of these bacteria, the anti-inflammation and/or gut barrier enhancer molecule will be detectable in the presence of RNS.

[0099] In certain embodiments, the anti-inflammation and/or gut barrier enhancer molecule is butyrate. Methods of measuring butyrate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 .mu.M/OD, at least about 10 .mu.M/OD, at least about 100 .mu.M/OD, at least about 500 .mu.M/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in the presence of RNS.

[0100] Secretion

[0101] In some embodiments, the genetically engineered bacteria further comprise a non-native secretion mechanism that is capable of secreting the anti-inflammation and/or gut barrier enhancer molecule from the bacterial cytoplasm. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm, injected into a target cell, or associated with the bacterial membrane.

[0102] In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system. Double membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Mycobacteria, which have a Gram-negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane-spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).

[0103] In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type III-like secretion system (T3SS) from Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the anti-inflammation and/or gut barrier enhancer molecule from the bacterial cytoplasm.

[0104] In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membrane-spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in E. coli), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the anti-inflammation and/or gut barrier enhancer molecule from the bacterial cytoplasm.

[0105] One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.

[0106] Treatment In Vivo

[0107] The genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with gut inflammation, compromised gut barrier function, and/or an autoimmune disorder may be used (see, e.g., Mizoguchi 2012). The animal model may be a mouse model of IBD, and IBD may be induced by treatment with dextran sodium sulfate. The animal model may be a mouse model of type 1 diabetes (T1D), and T1D may be induced by treatment with streptozotocin. In some embodiments, the genetically engineered bacteria of the invention is administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by endoscopy, colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. In some embodiments, the animal is sacrificed, and tissue samples are collected and analyzed, e.g., colonic sections are fixed and scored for inflammation and ulceration, and/or homogenized and analyzed for myeloperoxidase activity and cytokine levels (e.g., IL-1.beta., TNF-.alpha., IL-6, IFN-.gamma. and IL-10).

[0108] Essential Genes and Auxotrophs

[0109] As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).

[0110] An "essential gene" may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the recombinant bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.

[0111] An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria. For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0112] Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0113] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0114] In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

[0115] Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, foIC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, Igt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murI, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, IpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsI, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rpIW, rpID, rplC, rpsD, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rpIO, rpmD, rpsE, rpIR, rpIF, rpsH, rpsN, rpIE, rpIX, rpIN, rpsQ, rpmC, rpIP, rpsC, rpIV, rpsS, rpIB, cdsA, yaeL, yaeT, IpxD, fabZ, IpxA, IpxB, dnaE, accA, tiIS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, Int, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, IpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, loID, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, loIB, hemA, prfA, prmC, kdsA, topA, ribA, fabI, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

[0116] In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson "Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain," ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

[0117] In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, 13175, F319V, L340T, V3471, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, 13175, F319V, L340T, V3471, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L51 and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L51 and L6G.

[0118] In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, 13175, F319V, L340T, V3471, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

[0119] In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L). In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system.

[0120] In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the recombinant bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s). Other embodiments are described in Wright et al., "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (see Wright et al., supra).

[0121] Kill Switch

[0122] In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329 incorporated herein by reference in their entireties). The kill switch is intended to actively kill engineered microbes in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

[0123] Bacteria engineered with kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease or disorder may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a therapeutic gene(s) or after the subject has experienced the therapeutic effect. In some embodiments, the kill switch is activated to kill the bacteria after a period of time following RNS-mediated expression the anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the kill switch is activated in a delayed fashion following RNS-mediated expression the anti-inflammation and/or gut barrier function enhancer molecule. Alternatively, the bacteria may be engineered to die after the bacteria have spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject). Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl .beta.-D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010).

[0124] Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.

[0125] Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately lead to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable.

[0126] In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[0127] In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[0128] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.

[0129] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.

[0130] In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the wherein the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.

[0131] In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

[0132] In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxbl, PhiC31, TP901, Bxbl, PhiC31, TP901, HK022, HP1, R4, Int1, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, Int10, Int11, Int12, Int13, Int14, Int15, Int16, Int17, Int18, Int19, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof. In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill-switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present.

[0133] The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the AraBAD promoter, which induces expression of the desired gene.

[0134] Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an antitoxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

[0135] Arabinose inducible promoters are known in the art, including P.sub.ara, P.sub.araB, P.sub.araC, and P.sub.araBAD. In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the P.sub.araC promoter and the P.sub.araBAD promoter operate as a bidirectional promoter, with the P.sub.araBAD promoter controlling expression of a heterologous gene(s) in one direction, and the P.sub.araC (in close proximity to, and on the opposite strand from the P.sub.araBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

[0136] In one exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P.sub.araBAD promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a P.sub.araC promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (P.sub.TetR). In the presence of arabinose, the AraC transcription factor activates the P.sub.araBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the the P.sub.araBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the AraC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.

[0137] In one embodiment of the disclosure, the recombinant bacterial cell further comprises an antitoxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the antitoxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.

[0138] In another embodiment of the disclosure, the recombinant bacterial cell further comprises an antitoxin under the control of the P.sub.araBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.

[0139] In another exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P.sub.araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a P.sub.araC promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the P.sub.araBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the the P.sub.araBAD promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of P.sub.araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of P.sub.araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anto-toxin kill-switch system described directly above.

[0140] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FImA, Ibs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-051, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin E1, colicin K, colicin N, colicin U, colicin B, colicin Ia, colicin Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

[0141] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdID, Kis, SymR, MazE, FImB, Sib, ptaRNA1, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prIF, yefM, chpBl, hipB, MccE, MccE.sup.CTD, MccF, Cai, ImmE1, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

[0142] In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.

[0143] Mutagenesis

[0144] In some embodiments, a RNS-responsive regulatory region is operatively linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, the RNS-responsive regulatory region is mutagenized, and mutants are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence-activated cell sorting (FACS) when the detectable product fluoresces. In some embodiments, one or more transcription factor binding sites (see, e.g., sequences in FIGS. 4 and 5) are mutagenized to increase or decrease binding. In alternate embodiments, the wild-type binding sites are left in tact and the remainder of the regulatory region is subjected to mutagenesis. In some embodiments, the mutant regulatory region is inserted into the genetically engineered bacteria of the invention to increase expression of the anti-inflammation and/or gut barrier enhancer molecule in the presence of RNS, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the RNS-sensing transcription factor and/or the RNS-responsive regulatory region is a synthetic, non-naturally occurring sequence.

[0145] In some embodiments, the gene encoding an anti-inflammation and/or gut barrier enhancer molecule is mutated to increase expression and/or stability of said molecule in the presence of RNS, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, one or more of the genes in a gene cassette for producing an anti-inflammation and/or gut barrier enhancer molecule is mutated to increase expression of said molecule in the presence of RNS, as compared to unmutated bacteria of the same subtype under the same conditions.

[0146] Methods of Reporting Disease

[0147] In addition to producing therapeutic molecules, the genetically engineered bacteria of the invention may also be used to report disease pathology, progression, and/or resolution. Kotula et al. (2014) reported a binary toggle switch engineered into Escherichia coli to be induced in the gut of a mouse and remain stable in this configuration for at least seven days. These bacteria were able to sense a molecular event that flipped a toggle switch, which reported .beta.-galactosidase activity seven days later in stool samples. In some embodiments, the genetically engineered bacteria of the invention comprise a molecular switch, e.g., a toggle switch, a cis/trans riboregulator, or an adjustable threshold switch (Gardner et al., 2000; Callura et al., 2010; Kobayashi et al., 2004), for detecting a disease marker associated with gut inflammation and/or compromised gut barrier function, e.g., RNS, inflammatory cytokines, wherein the molecular switch is capable of producing a reporter, e.g., GFP, that is detected externally, e.g., in a stool sample, by endoscopy, in peripheral blood cells. In some embodiments, the reporter is expressed under the control of a RNS-responsive regulatory region. This molecular switch and reporter system may provide direct detection of molecular events and changes in disease pathology. In some embodiments, the molecular switch and reporter system is incorporated into the same bacteria that are delivering the anti-inflammation and/or gut barrier enhancer molecule. In alternate embodiments, the molecular switch and reporter system is incorporated into different bacteria than those delivering the anti-inflammation and/or gut barrier enhancer molecule. In some embodiments, methods using the molecular switch and reporter system may be used to develop therapies, screen for disease, stratify disease states, monitor disease progression, assess responsiveness to treatment, and/or to tailor and direct therapies.

[0148] Pharmaceutical Compositions and Formulations

[0149] Pharmaceutical compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent a disorder associated with hyperammonemia or symptom(s) associated with hyperammonemia. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[0150] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein.

[0151] The pharmaceutical compositions of the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[0152] The genetically engineered bacteria of the invention may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10.sup.5 to 10.sup.12 bacteria. The composition may be administered once or more daily, weekly, or monthly. The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents.

[0153] The genetically engineered bacteria of the invention may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

[0154] The genetically engineered bacteria of the invention may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[0155] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

[0156] In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

[0157] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria of the invention.

[0158] In certain embodiments, the genetically engineered bacteria of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[0159] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[0160] The genetically engineered bacteria of the invention may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0161] The genetically engineered bacteria of the invention may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0162] In some embodiments, the invention provides pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

[0163] Single dosage forms of the pharmaceutical composition of the invention may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[0164] Dosage regimens may be adjusted to provide a therapeutic response. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician.

[0165] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[0166] The genetically engineered bacteria of the invention may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0167] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0168] The pharmaceutical compositions of the invention may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions of the invention is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2.degree. C. and 8.degree. C. and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

[0169] Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD.sub.50, ED.sub.50, EC.sub.50, and IC.sub.50 may be determined, and the dose ratio between toxic and therapeutic effects (LD.sub.50/ED.sub.50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

[0170] Methods of Treatment

[0171] Another aspect of the invention provides methods of treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function. In some embodiments, the disease or condition is selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, intermediate colitis, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis. In some embodiments, the disease or condition is an autoimmune disorder 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, & Ill 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 (UP), 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. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to 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, and inflammation of the skin, eyes, joints, liver, and bile ducts. In some embodiments, the invention provides methods for reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing a systemic autoimmune disorder, e.g., asthma (Arrieta et al., 2015).

[0172] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g., by enema.

[0173] In certain embodiments, the pharmaceutical composition described herein is administered to reduce gut inflammation, enhance gut barrier function, and/or treat or prevent an autoimmune disorder in a subject. In some embodiments, the methods of the present disclosure may reduce gut inflammation in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, the methods of the present disclosure may enhance gut barrier function in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, changes in inflammation and/or gut barrier function are measured by comparing a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating the autoimmune disorder and/or the disease or condition associated with gut inflammation and/or compromised gut barrier function allows one or more symptoms of the disease or condition to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0174] Before, during, and after the administration of the pharmaceutical composition, gut inflammation and/or barrier function in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the invention to enhance gut barrier function and/or to reduce gut inflammation to baseline levels, e.g., levels comparable to those of a healthy control, in a subject. In some embodiments, the methods may include administration of the compositions of the invention to reduce gut inflammation to undetectable levels in a subject, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's levels prior to treatment. In some embodiments, the methods may include administration of the compositions of the invention to enhance gut barrier function in a subject by about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100% or more of the subject's levels prior to treatment.

[0175] In certain embodiments, the genetically engineered bacteria are E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the genetically engineered bacteria may be re-administered at a therapeutically effective dose and frequency. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

[0176] The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, e.g., corticosteroids, aminosalicylates, anti-inflammatory agents. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

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EXAMPLES

[0244] The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.

Example 1. Construction of Vectors for Overproducing Butyrate

[0245] To facilitate inducible production of butyrate in Escherichia coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk; NCBI; SEQ ID NO: 3-10), as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 to create pLogic031. The butyrate gene cassette was placed under control of a tetracycline-inducible promoter, with the tet repressor (TetR) expressed constitutively on another portion of the plasmid. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site.

[0246] The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co-oxidant. Because the recombinant bacteria of the invention are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from Treponema denticola, trans-2-enoynl-CoA reductase (ter), can functionally replace this three gene complex in an oxygen-independent manner. Therefore, we created a second plasmid capable of butyrate production in E. coli. Inverse PCR was used to amplify the entire sequence of pLogic031 outside of the bcd-etfA3-etfB3 region. The ter gene was codon optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt), synthesized (Genewiz, Cambridge, Mass.), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic046.

Example 2. Transforming E. coli with pLogic031 or pLogic046

[0247] The plasmid pLogic031 or pLogic046 was transformed into E. coli Nissle or E. coli DH5a. All tubes, solutions, and cuvettes are pre-chilled to 4.degree. C. An overnight culture of E. coli Nissle or E. coli DH5a was diluted 1:100 in 5 mL of lysogeny broth (LB) containing ampicillin and grown until it reached an OD.sub.600 of 0.4-0.6. The E. coli cells were then centrifuged at 2,000 rpm for 5 min. at 4.degree. C., the supernatant was removed, and the cells were resuspended in 1 mL of 4.degree. C. water. The E. coli were again centrifuged at 2,000 rpm for 5 min. at 4.degree. C., the supernatant was removed, and the cells were resuspended in 0.5 mL of 4.degree. C. water. The E. coli were again centrifuged at 2,000 rpm for 5 min. at 4.degree. C., the supernatant was removed, and the cells were finally resuspended in 0.1 mL of 4.degree. C. water. The electroporator was set to 2.5 kV. 0.5 .mu.g of one of the two pLogic plasmids was added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette was placed into the sample chamber, and the electric pulse was applied. One mL of room-temperature SOC media was immediately added, and the mixture is transferred to a culture tube and incubated at 37.degree. C. for 1 hr. The cells were spread out on an LB plate containing ampicillin and incubated overnight.

Example 3. Production of Butyrate in Recombinant E. coli

[0248] All incubations are performed at 37.degree. C. Cultures of E. coli strains DH5a and Nissle transformed with either pLogic031 or pLogic046 are grown overnight in LB and then diluted 1:50 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (200 rpm) for 2 h, at which time anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression the butyrate operon from the plasmids. Samples are collected at 2 h after addition of inducer for analysis of butyrate concentration by LC-MS.

[0249] Production of butyrate is assessed in E. coli Nissle strains containing pLogic031 and pLogic046 under microaerobic conditions in order to determine the effect of oxygen on butyrate production from these two plasmid variants. Overnight cultures are diluted 1:50 in M9 media containing 0.5% glucose and grown shaking (200 rpm) for 2 hours, at which point ATC is added to cultures (100 ng/mL). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.

Example 4. Construction of Vectors Encoding Butyrate Biosynthesis Cassette Under

[0250] Control of norB Regulatory Region

[0251] To create plasmids capable of nitric oxide-mediated induction of the butyrate operon, inverse PCR, using appropriate primers, is used to amplify the entire region of pLogic031 and pLogic046 outside of the tetR gene and tet promoter region. The nucleic acid sequence of pLogic031, comprising a tet promoter and butyrate operon (SEQ ID NO: 12), is shown in FIG. 6. The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are . The nucleic acid sequence of pLogic046, comprising a tet promoter and butyrate operon (SEQ ID NO: 13), is shown in FIG. 7. The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are . The nsrR gene and norB regulatory region from Neisseria gonorrhoeae are PCR amplified with overhangs homologous to the ends of these PCR fragments so that it may be inserted/cloned by Gibson assembly into the pLogic031 or pLogic046 derived PCR products. These constructs have tetracycline control region (tetR and tet promoter) replaced by the nsrR gene and norB regulatory region, which is induced by NsrR in the presence of NO. These newly assembled constructs, pLogic031-nsrR-norB and pLogic046-nsrR-norB, are used to transform E. coli Nissle through electroporation as described above, and transformants selected on ampicillin at 100 .mu.g/mL. Butyrate production from pLogic031-nsrR-norB and pLogic046-nsrR-norB in vitro is achieved by the addition of nitric oxide to cultures at 100 .mu.M. Butyrate levels in in vitro cultures are quantitated in culture supernatants by LC-MS as described above for the parent plasmids, pLogic031 and pLogic046.

Example 5. Construction of Vectors Encoding NsrR

[0252] Alternatively, nsrR may be expressed in a different plasmid than the one comprising the norB regulatory region and operatively linked gene(s) or gene cassette(s). The nsrR gene from Neisseria gonorrhoeae, which encodes the nitric oxide-sensitive protein responsible for driving expression from the norB regulatory region, is overexpressed from a medium copy plasmid in order to provide NsrR protein necessary for expression of the butyrate cassettes from pLogic031-norB or pLogic046-norB. The nsrR gene is cloned into the medium copy number plasmid under control of a constitutive promoter, such as P/ac, to create a plasmid that produces NsrR in a bacterium transformed therewith. This plasmid, which also bears an antibiotic resistance gene, such as kanamycin resistance, is used to transform E. coli Nissle already harboring pLogic031-norB or pLogic046-norB through electroporation. Transformants are selected in the presence of the antibiotic whose resistance is encoded in the plasmid (e.g., kanamycin at 50 .mu.g/mL) to select for those carrying the NsrR plasmid and ampicillin at 100 .mu.g/mL to maintain the plasmid already present (pLogic031-norB or pLogic046-norB). The resulting strains carry two plasmids, one containing the butyrate cassette, and the other containing the NsrR to control said cassette's induction. Butyrate production from these strains in vitro is achieved by the addition of nitric oxide to cultures at 100 .mu.M. Butyrate levels in culture supernatants are measured by LC-MS.

Example 6. Efficacy of Butyrate-Expressing Bacteria in a Mouse Model of IBD

[0253] Bacteria harboring both a plasmid expressing NsrR under control of a constitutive promoter and either pLogic031-nsrR-norB or pLogic046-nsrR-norB are grown overnight in LB supplemented with ampicillin. Bacteria are then diluted 1:100 into LB containing ampicillin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters is administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 3% dextran sodium sulfate for 7 days prior to bacterial gavage. Mice are treated daily for 1 week and bacteria in stool samples are detected by plating stool homogenate on agar plates supplemented with ampicillin. After 5 days of bacterial treatment, colitis is scored in live mice using endoscopy. Endoscopic damage score is determined by assessing colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. Mice are sacrificed and colonic tissues are isolated. Distal colonic sections are fixed and scored for inflammation and ulceration. Colonic tissue is homogenized and measurements are made for myeloperoxidase activity using an enzymatic assay kit and for cytokine levels (IL-1.beta., TNF-.alpha., IL-6, IFN-.gamma. and IL-10).

Example 7. Nitric Oxide-Inducible Reporter Constructs

[0254] ATC and nitric oxide-inducible reporter constructs were synthesized (Genewiz, Cambridge, Mass.). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations (FIGS. 12A-C); promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown in FIG. 13. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are .

Example 8. Nitric Oxide-Inducible Reporter Constructs in Mouse Model of IBD

[0255] Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1:100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) (FIG. 14). Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. FIG. 14 shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

Sequence CWU 1

1

1618575DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 1ttattatcgc accgcaatcg ggattttcga ttcataaagc aggtcgtagg tcggcttgtt 60gagcaggtct tgcagcgtga aaccgtccag atacgtgaaa aacgacttca ttgcaccgcc 120gagtatgccc gtcagccggc aggacggcgt aatcaggcat tcgttgttcg ggcccataca 180ctcgaccagc tgcatcggtt cgaggtggcg gacgaccgcg ccgatattga tgcgttcggg 240cggcgcggcc agcctcagcc cgccgccttt cccgcgtacg ctgtgcaaga acccgccttt 300gaccagcgcg gtaaccactt tcatcaaatg gcttttggaa atgccgtagg tcgaggcgat 360ggtggcgata ttgaccagcg cgtcgtcgtt gacggcggtg tagatgagga cgcgcagccc 420gtagtcggta tgttgggtca gatacataca acctccttag tacatgcaaa attatttcta 480gagcaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagttgagtt 540gaggaattat aacaggaaga aatattcctc atacgcttgt aattcctcta tggttgttga 600caattaatca tcggctcgta taatgtataa cattcatatt ttgtgaattt taaactctag 660aaataatttt gtttaacttt aagaaggaga tatacatatg gatttaaatt ctaaaaaata 720tcagatgctt aaagagctat atgtaagctt cgctgaaaat gaagttaaac ctttagcaac 780agaacttgat gaagaagaaa gatttcctta tgaaacagtg gaaaaaatgg caaaagcagg 840aatgatgggt ataccatatc caaaagaata tggtggagaa ggtggagaca ctgtaggata 900tataatggca gttgaagaat tgtctagagt ttgtggtact acaggagtta tattatcagc 960tcatacatct cttggctcat ggcctatata tcaatatggt aatgaagaac aaaaacaaaa 1020attcttaaga ccactagcaa gtggagaaaa attaggagca tttggtctta ctgagcctaa 1080tgctggtaca gatgcgtctg gccaacaaac aactgctgtt ttagacgggg atgaatacat 1140acttaatggc tcaaaaatat ttataacaaa cgcaatagct ggtgacatat atgtagtaat 1200ggcaatgact gataaatcta aggggaacaa aggaatatca gcatttatag ttgaaaaagg 1260aactcctggg tttagctttg gagttaaaga aaagaaaatg ggtataagag gttcagctac 1320gagtgaatta atatttgagg attgcagaat acctaaagaa aatttacttg gaaaagaagg 1380tcaaggattt aagatagcaa tgtctactct tgatggtggt agaattggta tagctgcaca 1440agctttaggt ttagcacaag gtgctcttga tgaaactgtt aaatatgtaa aagaaagagt 1500acaatttggt agaccattat caaaattcca aaatacacaa ttccaattag ctgatatgga 1560agttaaggta caagcggcta gacaccttgt atatcaagca gctataaata aagacttagg 1620aaaaccttat ggagtagaag cagcaatggc aaaattattt gcagctgaaa cagctatgga 1680agttactaca aaagctgtac aacttcatgg aggatatgga tacactcgtg actatccagt 1740agaaagaatg atgagagatg ctaagataac tgaaatatat gaaggaacta gtgaagttca 1800aagaatggtt atttcaggaa aactattaaa atagtaagaa ggagatatac atatggagga 1860aggatttatg aatatagtcg tttgtataaa acaagttcca gatacaacag aagttaaact 1920agatcctaat acaggtactt taattagaga tggagtacca agtataataa accctgatga 1980taaagcaggt ttagaagaag ctataaaatt aaaagaagaa atgggtgctc atgtaactgt 2040tataacaatg ggacctcctc aagcagatat ggctttaaaa gaagctttag caatgggtgc 2100agatagaggt atattattaa cagatagagc atttgcgggt gctgatactt gggcaacttc 2160atcagcatta gcaggagcat taaaaaatat agattttgat attataatag ctggaagaca 2220ggcgatagat ggagatactg cacaagttgg acctcaaata gctgaacatt taaatcttcc 2280atcaataaca tatgctgaag aaataaaaac tgaaggtgaa tatgtattag taaaaagaca 2340atttgaagat tgttgccatg acttaaaagt taaaatgcca tgccttataa caactcttaa 2400agatatgaac acaccaagat acatgaaagt tggaagaata tatgatgctt tcgaaaatga 2460tgtagtagaa acatggactg taaaagatat agaagttgac ccttctaatt taggtcttaa 2520aggttctcca actagtgtat ttaaatcatt tacaaaatca gttaaaccag ctggtacaat 2580atacaatgaa gatgcgaaaa catcagctgg aattatcata gataaattaa aagagaagta 2640tatcatataa taagaaggag atatacatat gggtaacgtt ttagtagtaa tagaacaaag 2700agaaaatgta attcaaactg tttctttaga attactagga aaggctacag aaatagcaaa 2760agattatgat acaaaagttt ctgcattact tttaggtagt aaggtagaag gtttaataga 2820tacattagca cactatggtg cagatgaggt aatagtagta gatgatgaag ctttagcagt 2880gtatacaact gaaccatata caaaagcagc ttatgaagca ataaaagcag ctgaccctat 2940agttgtatta tttggtgcaa cttcaatagg tagagattta gcgcctagag tttctgctag 3000aatacataca ggtcttactg ctgactgtac aggtcttgca gtagctgaag atacaaaatt 3060attattaatg acaagacctg cctttggtgg aaatataatg gcaacaatag tttgtaaaga 3120tttcagacct caaatgtcta cagttagacc aggggttatg aagaaaaatg aacctgatga 3180aactaaagaa gctgtaatta accgtttcaa ggtagaattt aatgatgctg ataaattagt 3240tcaagttgta caagtaataa aagaagctaa aaaacaagtt aaaatagaag atgctaagat 3300attagtttct gctggacgtg gaatgggtgg aaaagaaaac ttagacatac tttatgaatt 3360agctgaaatt ataggtggag aagtttctgg ttctcgtgcc actatagatg caggttggtt 3420agataaagca agacaagttg gtcaaactgg taaaactgta agaccagacc tttatatagc 3480atgtggtata tctggagcaa tacaacatat agctggtatg gaagatgctg agtttatagt 3540tgctataaat aaaaatccag aagctccaat atttaaatat gctgatgttg gtatagttgg 3600agatgttcat aaagtgcttc cagaacttat cagtcagtta agtgttgcaa aagaaaaagg 3660tgaagtttta gctaactaat aagaaggaga tatacatatg agagaagtag taattgccag 3720tgcagctaga acagcagtag gaagttttgg aggagcattt aaatcagttt cagcggtaga 3780gttaggggta acagcagcta aagaagctat aaaaagagct aacataactc cagatatgat 3840agatgaatct cttttagggg gagtacttac agcaggtctt ggacaaaata tagcaagaca 3900aatagcatta ggagcaggaa taccagtaga aaaaccagct atgactataa atatagtttg 3960tggttctgga ttaagatctg tttcaatggc atctcaactt atagcattag gtgatgctga 4020tataatgtta gttggtggag ctgaaaacat gagtatgtct ccttatttag taccaagtgc 4080gagatatggt gcaagaatgg gtgatgctgc ttttgttgat tcaatgataa aagatggatt 4140atcagacata tttaataact atcacatggg tattactgct gaaaacatag cagagcaatg 4200gaatataact agagaagaac aagatgaatt agctcttgca agtcaaaata aagctgaaaa 4260agctcaagct gaaggaaaat ttgatgaaga aatagttcct gttgttataa aaggaagaaa 4320aggtgacact gtagtagata aagatgaata tattaagcct ggcactacaa tggagaaact 4380tgctaagtta agacctgcat ttaaaaaaga tggaacagtt actgctggta atgcatcagg 4440aataaatgat ggtgctgcta tgttagtagt aatggctaaa gaaaaagctg aagaactagg 4500aatagagcct cttgcaacta tagtttctta tggaacagct ggtgttgacc ctaaaataat 4560gggatatgga ccagttccag caactaaaaa agctttagaa gctgctaata tgactattga 4620agatatagat ttagttgaag ctaatgaggc atttgctgcc caatctgtag ctgtaataag 4680agacttaaat atagatatga ataaagttaa tgttaatggt ggagcaatag ctataggaca 4740tccaatagga tgctcaggag caagaatact tactacactt ttatatgaaa tgaagagaag 4800agatgctaaa actggtcttg ctacactttg tataggcggt ggaatgggaa ctactttaat 4860agttaagaga tagtaagaag gagatataca tatgaaatta gctgtaatag gtagtggaac 4920tatgggaagt ggtattgtac aaacttttgc aagttgtgga catgatgtat gtttaaagag 4980tagaactcaa ggtgctatag ataaatgttt agctttatta gataaaaatt taactaagtt 5040agttactaag ggaaaaatgg atgaagctac aaaagcagaa atattaagtc atgttagttc 5100aactactaat tatgaagatt taaaagatat ggatttaata atagaagcat ctgtagaaga 5160catgaatata aagaaagatg ttttcaagtt actagatgaa ttatgtaaag aagatactat 5220cttggcaaca aatacttcat cattatctat aacagaaata gcttcttcta ctaagcgccc 5280agataaagtt ataggaatgc atttctttaa tccagttcct atgatgaaat tagttgaagt 5340tataagtggt cagttaacat caaaagttac ttttgataca gtatttgaat tatctaagag 5400tatcaataaa gtaccagtag atgtatctga atctcctgga tttgtagtaa atagaatact 5460tatacctatg ataaatgaag ctgttggtat atatgcagat ggtgttgcaa gtaaagaaga 5520aatagatgaa gctatgaaat taggagcaaa ccatccaatg ggaccactag cattaggtga 5580tttaatcgga ttagatgttg ttttagctat aatgaacgtt ttatatactg aatttggaga 5640tactaaatat agacctcatc cacttttagc taaaatggtt agagctaatc aattaggaag 5700aaaaactaag ataggattct atgattataa taaataataa gaaggagata tacatatgag 5760tacaagtgat gttaaagttt atgagaatgt agctgttgaa gtagatggaa atatatgtac 5820agtgaaaatg aatagaccta aagcccttaa tgcaataaat tcaaagactt tagaagaact 5880ttatgaagta tttgtagata ttaataatga tgaaactatt gatgttgtaa tattgacagg 5940ggaaggaaag gcatttgtag ctggagcaga tattgcatac atgaaagatt tagatgctgt 6000agctgctaaa gattttagta tcttaggagc aaaagctttt ggagaaatag aaaatagtaa 6060aaaagtagtg atagctgctg taaacggatt tgctttaggt ggaggatgtg aacttgcaat 6120ggcatgtgat ataagaattg catctgctaa agctaaattt ggtcagccag aagtaactct 6180tggaataact ccaggatatg gaggaactca aaggcttaca agattggttg gaatggcaaa 6240agcaaaagaa ttaatcttta caggtcaagt tataaaagct gatgaagctg aaaaaatagg 6300gctagtaaat agagtcgttg agccagacat tttaatagaa gaagttgaga aattagctaa 6360gataatagct aaaaatgctc agcttgcagt tagatactct aaagaagcaa tacaacttgg 6420tgctcaaact gatataaata ctggaataga tatagaatct aatttatttg gtctttgttt 6480ttcaactaaa gaccaaaaag aaggaatgtc agctttcgtt gaaaagagag aagctaactt 6540tataaaaggg taataagaag gagatataca tatgagaagt tttgaagaag taattaagtt 6600tgcaaaagaa agaggaccta aaactatatc agtagcatgt tgccaagata aagaagtttt 6660aatggcagtt gaaatggcta gaaaagaaaa aatagcaaat gccattttag taggagatat 6720agaaaagact aaagaaattg caaaaagcat agacatggat atcgaaaatt atgaactgat 6780agatataaaa gatttagcag aagcatctct aaaatctgtt gaattagttt cacaaggaaa 6840agccgacatg gtaatgaaag gcttagtaga cacatcaata atactaaaag cagttttaaa 6900taaagaagta ggtcttagaa ctggaaatgt attaagtcac gtagcagtat ttgatgtaga 6960gggatatgat agattatttt tcgtaactga cgcagctatg aacttagctc ctgatacaaa 7020tactaaaaag caaatcatag aaaatgcttg cacagtagca cattcattag atataagtga 7080accaaaagtt gctgcaatat gcgcaaaaga aaaagtaaat ccaaaaatga aagatacagt 7140tgaagctaaa gaactagaag aaatgtatga aagaggagaa atcaaaggtt gtatggttgg 7200tgggcctttt gcaattgata atgcagtatc tttagaagca gctaaacata aaggtataaa 7260tcatcctgta gcaggacgag ctgatatatt attagcccca gatattgaag gtggtaacat 7320attatataaa gctttggtat tcttctcaaa atcaaaaaat gcaggagtta tagttggggc 7380taaagcacca ataatattaa cttctagagc agacagtgaa gaaactaaac taaactcaat 7440agctttaggt gttttaatgg cagcaaaggc ataataagaa ggagatatac atatgagcaa 7500aatatttaaa atcttaacaa taaatcctgg ttcgacatca actaaaatag ctgtatttga 7560taatgaggat ttagtatttg aaaaaacttt aagacattct tcagaagaaa taggaaaata 7620tgagaaggtg tctgaccaat ttgaatttcg taaacaagta atagaagaag ctctaaaaga 7680aggtggagta aaaacatctg aattagatgc tgtagtaggt agaggaggac ttcttaaacc 7740tataaaaggt ggtacttatt cagtaagtgc tgctatgatt gaagatttaa aagtgggagt 7800tttaggagaa cacgcttcaa acctaggtgg aataatagca aaacaaatag gtgaagaagt 7860aaatgttcct tcatacatag tagaccctgt tgttgtagat gaattagaag atgttgctag 7920aatttctggt atgcctgaaa taagtagagc aagtgtagta catgctttaa atcaaaaggc 7980aatagcaaga agatatgcta gagaaataaa caagaaatat gaagatataa atcttatagt 8040tgcacacatg ggtggaggag tttctgttgg agctcataaa aatggtaaaa tagtagatgt 8100tgcaaacgca ttagatggag aaggaccttt ctctccagaa agaagtggtg gactaccagt 8160aggtgcatta gtaaaaatgt gctttagtgg aaaatatact caagatgaaa ttaaaaagaa 8220aataaaaggt aatggcggac tagttgcata cttaaacact aatgatgcta gagaagttga 8280agaaagaatt gaagctggtg atgaaaaagc taaattagta tatgaagcta tggcatatca 8340aatctctaaa gaaataggag ctagtgctgc agttcttaag ggagatgtaa aagcaatatt 8400attaactggt ggaatcgcat attcaaaaat gtttacagaa atgattgcag atagagttaa 8460atttatagca gatgtaaaag tttatccagg tgaagatgaa atgattgcat tagctcaagg 8520tggacttaga gttttaactg gtgaagaaga ggctcaagtt tatgataact aataa 857526787DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 2ttattatcgc accgcaatcg ggattttcga ttcataaagc aggtcgtagg tcggcttgtt 60gagcaggtct tgcagcgtga aaccgtccag atacgtgaaa aacgacttca ttgcaccgcc 120gagtatgccc gtcagccggc aggacggcgt aatcaggcat tcgttgttcg ggcccataca 180ctcgaccagc tgcatcggtt cgaggtggcg gacgaccgcg ccgatattga tgcgttcggg 240cggcgcggcc agcctcagcc cgccgccttt cccgcgtacg ctgtgcaaga acccgccttt 300gaccagcgcg gtaaccactt tcatcaaatg gcttttggaa atgccgtagg tcgaggcgat 360ggtggcgata ttgaccagcg cgtcgtcgtt gacggcggtg tagatgagga cgcgcagccc 420gtagtcggta tgttgggtca gatacataca acctccttag tacatgcaaa attatttcta 480gagcaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagttgagtt 540gaggaattat aacaggaaga aatattcctc atacgcttgt aattcctcta tggttgttga 600caattaatca tcggctcgta taatgtataa cattcatatt ttgtgaattt taaactctag 660aaataatttt gtttaacttt aagaaggaga tatacatatg atcgtaaaac ctatggtacg 720caacaatatc tgcctgaacg cccatcctca gggctgcaag aagggagtgg aagatcagat 780tgaatatacc aagaaacgca ttaccgcaga agtcaaagct ggcgcaaaag ctccaaaaaa 840cgttctggtg cttggctgct caaatggtta cggcctggcg agccgcatta ctgctgcgtt 900cggatacggg gctgcgacca tcggcgtgtc ctttgaaaaa gcgggttcag aaaccaaata 960tggtacaccg ggatggtaca ataatttggc atttgatgaa gcggcaaaac gcgagggtct 1020ttatagcgtg acgatcgacg gcgatgcgtt ttcagacgag atcaaggccc aggtaattga 1080ggaagccaaa aaaaaaggta tcaaatttga tctgatcgta tacagcttgg ccagcccagt 1140acgtactgat cctgatacag gtatcatgca caaaagcgtt ttgaaaccct ttggaaaaac 1200gttcacaggc aaaacagtag atccgtttac tggcgagctg aaggaaatct ccgcggaacc 1260agcaaatgac gaggaagcag ccgccactgt taaagttatg gggggtgaag attgggaacg 1320ttggattaag cagctgtcga aggaaggcct cttagaagaa ggctgtatta ccttggccta 1380tagttatatt ggccctgaag ctacccaagc tttgtaccgt aaaggcacaa tcggcaaggc 1440caaagaacac ctggaggcca cagcacaccg tctcaacaaa gagaacccgt caatccgtgc 1500cttcgtgagc gtgaataaag gcctggtaac ccgcgcaagc gccgtaatcc cggtaatccc 1560tctgtatctc gccagcttgt tcaaagtaat gaaagagaag ggcaatcatg aaggttgtat 1620tgaacagatc acgcgtctgt acgccgagcg cctgtaccgt aaagatggta caattccagt 1680tgatgaggaa aatcgcattc gcattgatga ttgggagtta gaagaagacg tccagaaagc 1740ggtatccgcg ttgatggaga aagtcacggg tgaaaacgca gaatctctca ctgacttagc 1800ggggtaccgc catgatttct tagctagtaa cggctttgat gtagaaggta ttaattatga 1860agcggaagtt gaacgcttcg accgtatctg ataagaagga gatatacata tgagagaagt 1920agtaattgcc agtgcagcta gaacagcagt aggaagtttt ggaggagcat ttaaatcagt 1980ttcagcggta gagttagggg taacagcagc taaagaagct ataaaaagag ctaacataac 2040tccagatatg atagatgaat ctcttttagg gggagtactt acagcaggtc ttggacaaaa 2100tatagcaaga caaatagcat taggagcagg aataccagta gaaaaaccag ctatgactat 2160aaatatagtt tgtggttctg gattaagatc tgtttcaatg gcatctcaac ttatagcatt 2220aggtgatgct gatataatgt tagttggtgg agctgaaaac atgagtatgt ctccttattt 2280agtaccaagt gcgagatatg gtgcaagaat gggtgatgct gcttttgttg attcaatgat 2340aaaagatgga ttatcagaca tatttaataa ctatcacatg ggtattactg ctgaaaacat 2400agcagagcaa tggaatataa ctagagaaga acaagatgaa ttagctcttg caagtcaaaa 2460taaagctgaa aaagctcaag ctgaaggaaa atttgatgaa gaaatagttc ctgttgttat 2520aaaaggaaga aaaggtgaca ctgtagtaga taaagatgaa tatattaagc ctggcactac 2580aatggagaaa cttgctaagt taagacctgc atttaaaaaa gatggaacag ttactgctgg 2640taatgcatca ggaataaatg atggtgctgc tatgttagta gtaatggcta aagaaaaagc 2700tgaagaacta ggaatagagc ctcttgcaac tatagtttct tatggaacag ctggtgttga 2760ccctaaaata atgggatatg gaccagttcc agcaactaaa aaagctttag aagctgctaa 2820tatgactatt gaagatatag atttagttga agctaatgag gcatttgctg cccaatctgt 2880agctgtaata agagacttaa atatagatat gaataaagtt aatgttaatg gtggagcaat 2940agctatagga catccaatag gatgctcagg agcaagaata cttactacac ttttatatga 3000aatgaagaga agagatgcta aaactggtct tgctacactt tgtataggcg gtggaatggg 3060aactacttta atagttaaga gatagtaaga aggagatata catatgaaat tagctgtaat 3120aggtagtgga actatgggaa gtggtattgt acaaactttt gcaagttgtg gacatgatgt 3180atgtttaaag agtagaactc aaggtgctat agataaatgt ttagctttat tagataaaaa 3240tttaactaag ttagttacta agggaaaaat ggatgaagct acaaaagcag aaatattaag 3300tcatgttagt tcaactacta attatgaaga tttaaaagat atggatttaa taatagaagc 3360atctgtagaa gacatgaata taaagaaaga tgttttcaag ttactagatg aattatgtaa 3420agaagatact atcttggcaa caaatacttc atcattatct ataacagaaa tagcttcttc 3480tactaagcgc ccagataaag ttataggaat gcatttcttt aatccagttc ctatgatgaa 3540attagttgaa gttataagtg gtcagttaac atcaaaagtt acttttgata cagtatttga 3600attatctaag agtatcaata aagtaccagt agatgtatct gaatctcctg gatttgtagt 3660aaatagaata cttataccta tgataaatga agctgttggt atatatgcag atggtgttgc 3720aagtaaagaa gaaatagatg aagctatgaa attaggagca aaccatccaa tgggaccact 3780agcattaggt gatttaatcg gattagatgt tgttttagct ataatgaacg ttttatatac 3840tgaatttgga gatactaaat atagacctca tccactttta gctaaaatgg ttagagctaa 3900tcaattagga agaaaaacta agataggatt ctatgattat aataaataat aagaaggaga 3960tatacatatg agtacaagtg atgttaaagt ttatgagaat gtagctgttg aagtagatgg 4020aaatatatgt acagtgaaaa tgaatagacc taaagccctt aatgcaataa attcaaagac 4080tttagaagaa ctttatgaag tatttgtaga tattaataat gatgaaacta ttgatgttgt 4140aatattgaca ggggaaggaa aggcatttgt agctggagca gatattgcat acatgaaaga 4200tttagatgct gtagctgcta aagattttag tatcttagga gcaaaagctt ttggagaaat 4260agaaaatagt aaaaaagtag tgatagctgc tgtaaacgga tttgctttag gtggaggatg 4320tgaacttgca atggcatgtg atataagaat tgcatctgct aaagctaaat ttggtcagcc 4380agaagtaact cttggaataa ctccaggata tggaggaact caaaggctta caagattggt 4440tggaatggca aaagcaaaag aattaatctt tacaggtcaa gttataaaag ctgatgaagc 4500tgaaaaaata gggctagtaa atagagtcgt tgagccagac attttaatag aagaagttga 4560gaaattagct aagataatag ctaaaaatgc tcagcttgca gttagatact ctaaagaagc 4620aatacaactt ggtgctcaaa ctgatataaa tactggaata gatatagaat ctaatttatt 4680tggtctttgt ttttcaacta aagaccaaaa agaaggaatg tcagctttcg ttgaaaagag 4740agaagctaac tttataaaag ggtaataaga aggagatata catatgagaa gttttgaaga 4800agtaattaag tttgcaaaag aaagaggacc taaaactata tcagtagcat gttgccaaga 4860taaagaagtt ttaatggcag ttgaaatggc tagaaaagaa aaaatagcaa atgccatttt 4920agtaggagat atagaaaaga ctaaagaaat tgcaaaaagc atagacatgg atatcgaaaa 4980ttatgaactg atagatataa aagatttagc agaagcatct ctaaaatctg ttgaattagt 5040ttcacaagga aaagccgaca tggtaatgaa aggcttagta gacacatcaa taatactaaa 5100agcagtttta aataaagaag taggtcttag aactggaaat gtattaagtc acgtagcagt 5160atttgatgta gagggatatg atagattatt tttcgtaact gacgcagcta tgaacttagc 5220tcctgataca aatactaaaa agcaaatcat agaaaatgct tgcacagtag cacattcatt 5280agatataagt gaaccaaaag ttgctgcaat atgcgcaaaa gaaaaagtaa atccaaaaat 5340gaaagataca gttgaagcta aagaactaga agaaatgtat gaaagaggag aaatcaaagg 5400ttgtatggtt ggtgggcctt ttgcaattga taatgcagta tctttagaag cagctaaaca 5460taaaggtata aatcatcctg tagcaggacg agctgatata ttattagccc cagatattga 5520aggtggtaac atattatata aagctttggt attcttctca aaatcaaaaa atgcaggagt 5580tatagttggg gctaaagcac caataatatt aacttctaga gcagacagtg aagaaactaa 5640actaaactca atagctttag gtgttttaat ggcagcaaag gcataataag aaggagatat 5700acatatgagc aaaatattta aaatcttaac aataaatcct ggttcgacat caactaaaat 5760agctgtattt gataatgagg atttagtatt tgaaaaaact ttaagacatt cttcagaaga 5820aataggaaaa tatgagaagg tgtctgacca atttgaattt cgtaaacaag taatagaaga 5880agctctaaaa gaaggtggag taaaaacatc tgaattagat gctgtagtag gtagaggagg 5940acttcttaaa cctataaaag gtggtactta ttcagtaagt gctgctatga ttgaagattt 6000aaaagtggga gttttaggag aacacgcttc aaacctaggt ggaataatag caaaacaaat 6060aggtgaagaa gtaaatgttc cttcatacat agtagaccct gttgttgtag atgaattaga 6120agatgttgct agaatttctg gtatgcctga aataagtaga gcaagtgtag tacatgcttt 6180aaatcaaaag gcaatagcaa gaagatatgc tagagaaata aacaagaaat atgaagatat 6240aaatcttata gttgcacaca tgggtggagg agtttctgtt ggagctcata aaaatggtaa 6300aatagtagat gttgcaaacg

cattagatgg agaaggacct ttctctccag aaagaagtgg 6360tggactacca gtaggtgcat tagtaaaaat gtgctttagt ggaaaatata ctcaagatga 6420aattaaaaag aaaataaaag gtaatggcgg actagttgca tacttaaaca ctaatgatgc 6480tagagaagtt gaagaaagaa ttgaagctgg tgatgaaaaa gctaaattag tatatgaagc 6540tatggcatat caaatctcta aagaaatagg agctagtgct gcagttctta agggagatgt 6600aaaagcaata ttattaactg gtggaatcgc atattcaaaa atgtttacag aaatgattgc 6660agatagagtt aaatttatag cagatgtaaa agtttatcca ggtgaagatg aaatgattgc 6720attagctcaa ggtggactta gagttttaac tggtgaagaa gaggctcaag tttatgataa 6780ctaataa 678731137DNAPeptoclostridium difficile 3atggatttaa attctaaaaa atatcagatg cttaaagagc tatatgtaag cttcgctgaa 60aatgaagtta aacctttagc aacagaactt gatgaagaag aaagatttcc ttatgaaaca 120gtggaaaaaa tggcaaaagc aggaatgatg ggtataccat atccaaaaga atatggtgga 180gaaggtggag acactgtagg atatataatg gcagttgaag aattgtctag agtttgtggt 240actacaggag ttatattatc agctcataca tctcttggct catggcctat atatcaatat 300ggtaatgaag aacaaaaaca aaaattctta agaccactag caagtggaga aaaattagga 360gcatttggtc ttactgagcc taatgctggt acagatgcgt ctggccaaca aacaactgct 420gttttagacg gggatgaata catacttaat ggctcaaaaa tatttataac aaacgcaata 480gctggtgaca tatatgtagt aatggcaatg actgataaat ctaaggggaa caaaggaata 540tcagcattta tagttgaaaa aggaactcct gggtttagct ttggagttaa agaaaagaaa 600atgggtataa gaggttcagc tacgagtgaa ttaatatttg aggattgcag aatacctaaa 660gaaaatttac ttggaaaaga aggtcaagga tttaagatag caatgtctac tcttgatggt 720ggtagaattg gtatagctgc acaagcttta ggtttagcac aaggtgctct tgatgaaact 780gttaaatatg taaaagaaag agtacaattt ggtagaccat tatcaaaatt ccaaaataca 840caattccaat tagctgatat ggaagttaag gtacaagcgg ctagacacct tgtatatcaa 900gcagctataa ataaagactt aggaaaacct tatggagtag aagcagcaat ggcaaaatta 960tttgcagctg aaacagctat ggaagttact acaaaagctg tacaacttca tggaggatat 1020ggatacactc gtgactatcc agtagaaaga atgatgagag atgctaagat aactgaaata 1080tatgaaggaa ctagtgaagt tcaaagaatg gttatttcag gaaaactatt aaaatag 11374783DNAPeptoclostridium difficile 4atgaatatag tcgtttgtat aaaacaagtt ccagatacaa cagaagttaa actagatcct 60aatacaggta ctttaattag agatggagta ccaagtataa taaaccctga tgataaagca 120ggtttagaag aagctataaa attaaaagaa gaaatgggtg ctcatgtaac tgttataaca 180atgggacctc ctcaagcaga tatggcttta aaagaagctt tagcaatggg tgcagataga 240ggtatattat taacagatag agcatttgcg ggtgctgata cttgggcaac ttcatcagca 300ttagcaggag cattaaaaaa tatagatttt gatattataa tagctggaag acaggcgata 360gatggagata ctgcacaagt tggacctcaa atagctgaac atttaaatct tccatcaata 420acatatgctg aagaaataaa aactgaaggt gaatatgtat tagtaaaaag acaatttgaa 480gattgttgcc atgacttaaa agttaaaatg ccatgcctta taacaactct taaagatatg 540aacacaccaa gatacatgaa agttggaaga atatatgatg ctttcgaaaa tgatgtagta 600gaaacatgga ctgtaaaaga tatagaagtt gacccttcta atttaggtct taaaggttct 660ccaactagtg tatttaaatc atttacaaaa tcagttaaac cagctggtac aatatacaat 720gaagatgcga aaacatcagc tggaattatc atagataaat taaaagagaa gtatatcata 780taa 78351011DNAPeptoclostridium difficile 5atgggtaacg ttttagtagt aatagaacaa agagaaaatg taattcaaac tgtttcttta 60gaattactag gaaaggctac agaaatagca aaagattatg atacaaaagt ttctgcatta 120cttttaggta gtaaggtaga aggtttaata gatacattag cacactatgg tgcagatgag 180gtaatagtag tagatgatga agctttagca gtgtatacaa ctgaaccata tacaaaagca 240gcttatgaag caataaaagc agctgaccct atagttgtat tatttggtgc aacttcaata 300ggtagagatt tagcgcctag agtttctgct agaatacata caggtcttac tgctgactgt 360acaggtcttg cagtagctga agatacaaaa ttattattaa tgacaagacc tgcctttggt 420ggaaatataa tggcaacaat agtttgtaaa gatttcagac ctcaaatgtc tacagttaga 480ccaggggtta tgaagaaaaa tgaacctgat gaaactaaag aagctgtaat taaccgtttc 540aaggtagaat ttaatgatgc tgataaatta gttcaagttg tacaagtaat aaaagaagct 600aaaaaacaag ttaaaataga agatgctaag atattagttt ctgctggacg tggaatgggt 660ggaaaagaaa acttagacat actttatgaa ttagctgaaa ttataggtgg agaagtttct 720ggttctcgtg ccactataga tgcaggttgg ttagataaag caagacaagt tggtcaaact 780ggtaaaactg taagaccaga cctttatata gcatgtggta tatctggagc aatacaacat 840atagctggta tggaagatgc tgagtttata gttgctataa ataaaaatcc agaagctcca 900atatttaaat atgctgatgt tggtatagtt ggagatgttc ataaagtgct tccagaactt 960atcagtcagt taagtgttgc aaaagaaaaa ggtgaagttt tagctaacta a 101161176DNAPeptoclostridium difficile 6atgagagaag tagtaattgc cagtgcagct agaacagcag taggaagttt tggaggagca 60tttaaatcag tttcagcggt agagttaggg gtaacagcag ctaaagaagc tataaaaaga 120gctaacataa ctccagatat gatagatgaa tctcttttag ggggagtact tacagcaggt 180cttggacaaa atatagcaag acaaatagca ttaggagcag gaataccagt agaaaaacca 240gctatgacta taaatatagt ttgtggttct ggattaagat ctgtttcaat ggcatctcaa 300cttatagcat taggtgatgc tgatataatg ttagttggtg gagctgaaaa catgagtatg 360tctccttatt tagtaccaag tgcgagatat ggtgcaagaa tgggtgatgc tgcttttgtt 420gattcaatga taaaagatgg attatcagac atatttaata actatcacat gggtattact 480gctgaaaaca tagcagagca atggaatata actagagaag aacaagatga attagctctt 540gcaagtcaaa ataaagctga aaaagctcaa gctgaaggaa aatttgatga agaaatagtt 600cctgttgtta taaaaggaag aaaaggtgac actgtagtag ataaagatga atatattaag 660cctggcacta caatggagaa acttgctaag ttaagacctg catttaaaaa agatggaaca 720gttactgctg gtaatgcatc aggaataaat gatggtgctg ctatgttagt agtaatggct 780aaagaaaaag ctgaagaact aggaatagag cctcttgcaa ctatagtttc ttatggaaca 840gctggtgttg accctaaaat aatgggatat ggaccagttc cagcaactaa aaaagcttta 900gaagctgcta atatgactat tgaagatata gatttagttg aagctaatga ggcatttgct 960gcccaatctg tagctgtaat aagagactta aatatagata tgaataaagt taatgttaat 1020ggtggagcaa tagctatagg acatccaata ggatgctcag gagcaagaat acttactaca 1080cttttatatg aaatgaagag aagagatgct aaaactggtc ttgctacact ttgtataggc 1140ggtggaatgg gaactacttt aatagttaag agatag 11767846DNAPeptoclostridium difficile 7atgaaattag ctgtaatagg tagtggaact atgggaagtg gtattgtaca aacttttgca 60agttgtggac atgatgtatg tttaaagagt agaactcaag gtgctataga taaatgttta 120gctttattag ataaaaattt aactaagtta gttactaagg gaaaaatgga tgaagctaca 180aaagcagaaa tattaagtca tgttagttca actactaatt atgaagattt aaaagatatg 240gatttaataa tagaagcatc tgtagaagac atgaatataa agaaagatgt tttcaagtta 300ctagatgaat tatgtaaaga agatactatc ttggcaacaa atacttcatc attatctata 360acagaaatag cttcttctac taagcgccca gataaagtta taggaatgca tttctttaat 420ccagttccta tgatgaaatt agttgaagtt ataagtggtc agttaacatc aaaagttact 480tttgatacag tatttgaatt atctaagagt atcaataaag taccagtaga tgtatctgaa 540tctcctggat ttgtagtaaa tagaatactt atacctatga taaatgaagc tgttggtata 600tatgcagatg gtgttgcaag taaagaagaa atagatgaag ctatgaaatt aggagcaaac 660catccaatgg gaccactagc attaggtgat ttaatcggat tagatgttgt tttagctata 720atgaacgttt tatatactga atttggagat actaaatata gacctcatcc acttttagct 780aaaatggtta gagctaatca attaggaaga aaaactaaga taggattcta tgattataat 840aaataa 8468798DNAPeptoclostridium difficile 8atgagtacaa gtgatgttaa agtttatgag aatgtagctg ttgaagtaga tggaaatata 60tgtacagtga aaatgaatag acctaaagcc cttaatgcaa taaattcaaa gactttagaa 120gaactttatg aagtatttgt agatattaat aatgatgaaa ctattgatgt tgtaatattg 180acaggggaag gaaaggcatt tgtagctgga gcagatattg catacatgaa agatttagat 240gctgtagctg ctaaagattt tagtatctta ggagcaaaag cttttggaga aatagaaaat 300agtaaaaaag tagtgatagc tgctgtaaac ggatttgctt taggtggagg atgtgaactt 360gcaatggcat gtgatataag aattgcatct gctaaagcta aatttggtca gccagaagta 420actcttggaa taactccagg atatggagga actcaaaggc ttacaagatt ggttggaatg 480gcaaaagcaa aagaattaat ctttacaggt caagttataa aagctgatga agctgaaaaa 540atagggctag taaatagagt cgttgagcca gacattttaa tagaagaagt tgagaaatta 600gctaagataa tagctaaaaa tgctcagctt gcagttagat actctaaaga agcaatacaa 660cttggtgctc aaactgatat aaatactgga atagatatag aatctaattt atttggtctt 720tgtttttcaa ctaaagacca aaaagaagga atgtcagctt tcgttgaaaa gagagaagct 780aactttataa aagggtaa 7989903DNAPeptoclostridium difficile 9atgagaagtt ttgaagaagt aattaagttt gcaaaagaaa gaggacctaa aactatatca 60gtagcatgtt gccaagataa agaagtttta atggcagttg aaatggctag aaaagaaaaa 120atagcaaatg ccattttagt aggagatata gaaaagacta aagaaattgc aaaaagcata 180gacatggata tcgaaaatta tgaactgata gatataaaag atttagcaga agcatctcta 240aaatctgttg aattagtttc acaaggaaaa gccgacatgg taatgaaagg cttagtagac 300acatcaataa tactaaaagc agttttaaat aaagaagtag gtcttagaac tggaaatgta 360ttaagtcacg tagcagtatt tgatgtagag ggatatgata gattattttt cgtaactgac 420gcagctatga acttagctcc tgatacaaat actaaaaagc aaatcataga aaatgcttgc 480acagtagcac attcattaga tataagtgaa ccaaaagttg ctgcaatatg cgcaaaagaa 540aaagtaaatc caaaaatgaa agatacagtt gaagctaaag aactagaaga aatgtatgaa 600agaggagaaa tcaaaggttg tatggttggt gggccttttg caattgataa tgcagtatct 660ttagaagcag ctaaacataa aggtataaat catcctgtag caggacgagc tgatatatta 720ttagccccag atattgaagg tggtaacata ttatataaag ctttggtatt cttctcaaaa 780tcaaaaaatg caggagttat agttggggct aaagcaccaa taatattaac ttctagagca 840gacagtgaag aaactaaact aaactcaata gctttaggtg ttttaatggc agcaaaggca 900taa 903101080DNAPeptoclostridium difficile 10atgagcaaaa tatttaaaat cttaacaata aatcctggtt cgacatcaac taaaatagct 60gtatttgata atgaggattt agtatttgaa aaaactttaa gacattcttc agaagaaata 120ggaaaatatg agaaggtgtc tgaccaattt gaatttcgta aacaagtaat agaagaagct 180ctaaaagaag gtggagtaaa aacatctgaa ttagatgctg tagtaggtag aggaggactt 240cttaaaccta taaaaggtgg tacttattca gtaagtgctg ctatgattga agatttaaaa 300gtgggagttt taggagaaca cgcttcaaac ctaggtggaa taatagcaaa acaaataggt 360gaagaagtaa atgttccttc atacatagta gaccctgttg ttgtagatga attagaagat 420gttgctagaa tttctggtat gcctgaaata agtagagcaa gtgtagtaca tgctttaaat 480caaaaggcaa tagcaagaag atatgctaga gaaataaaca agaaatatga agatataaat 540cttatagttg cacacatggg tggaggagtt tctgttggag ctcataaaaa tggtaaaata 600gtagatgttg caaacgcatt agatggagaa ggacctttct ctccagaaag aagtggtgga 660ctaccagtag gtgcattagt aaaaatgtgc tttagtggaa aatatactca agatgaaatt 720aaaaagaaaa taaaaggtaa tggcggacta gttgcatact taaacactaa tgatgctaga 780gaagttgaag aaagaattga agctggtgat gaaaaagcta aattagtata tgaagctatg 840gcatatcaaa tctctaaaga aataggagct agtgctgcag ttcttaaggg agatgtaaaa 900gcaatattat taactggtgg aatcgcatat tcaaaaatgt ttacagaaat gattgcagat 960agagttaaat ttatagcaga tgtaaaagtt tatccaggtg aagatgaaat gattgcatta 1020gctcaaggtg gacttagagt tttaactggt gaagaagagg ctcaagttta tgataactaa 1080111194DNATreponema denticola 11atgatcgtaa aacctatggt acgcaacaat atctgcctga acgcccatcc tcagggctgc 60aagaagggag tggaagatca gattgaatat accaagaaac gcattaccgc agaagtcaaa 120gctggcgcaa aagctccaaa aaacgttctg gtgcttggct gctcaaatgg ttacggcctg 180gcgagccgca ttactgctgc gttcggatac ggggctgcga ccatcggcgt gtcctttgaa 240aaagcgggtt cagaaaccaa atatggtaca ccgggatggt acaataattt ggcatttgat 300gaagcggcaa aacgcgaggg tctttatagc gtgacgatcg acggcgatgc gttttcagac 360gagatcaagg cccaggtaat tgaggaagcc aaaaaaaaag gtatcaaatt tgatctgatc 420gtatacagct tggccagccc agtacgtact gatcctgata caggtatcat gcacaaaagc 480gttttgaaac cctttggaaa aacgttcaca ggcaaaacag tagatccgtt tactggcgag 540ctgaaggaaa tctccgcgga accagcaaat gacgaggaag cagccgccac tgttaaagtt 600atggggggtg aagattggga acgttggatt aagcagctgt cgaaggaagg cctcttagaa 660gaaggctgta ttaccttggc ctatagttat attggccctg aagctaccca agctttgtac 720cgtaaaggca caatcggcaa ggccaaagaa cacctggagg ccacagcaca ccgtctcaac 780aaagagaacc cgtcaatccg tgccttcgtg agcgtgaata aaggcctggt aacccgcgca 840agcgccgtaa tcccggtaat ccctctgtat ctcgccagct tgttcaaagt aatgaaagag 900aagggcaatc atgaaggttg tattgaacag atcacgcgtc tgtacgccga gcgcctgtac 960cgtaaagatg gtacaattcc agttgatgag gaaaatcgca ttcgcattga tgattgggag 1020ttagaagaag acgtccagaa agcggtatcc gcgttgatgg agaaagtcac gggtgaaaac 1080gcagaatctc tcactgactt agcggggtac cgccatgatt tcttagctag taacggcttt 1140gatgtagaag gtattaatta tgaagcggaa gttgaacgct tcgaccgtat ctga 1194128650DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 12gtaaaacgac ggccagtgaa ttcgttaaga cccactttca catttaagtt gtttttctaa 60tccgcatatg atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa 120taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc 180ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac 240agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa 300ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat gtacttttgc 360tccatcgcga tgacttagta aagcacatct aaaactttta gcgttattac gtaaaaaatc 420ttgccagctt tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat 480ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc 540tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag 600cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatca ttaattccta 660atttttgttg acactctatc attgatagag ttattttacc actccctatc agtgatagag 720aaaagtgaac tctagaaata attttgttta actttaagaa ggagatatac atatggattt 780aaattctaaa aaatatcaga tgcttaaaga gctatatgta agcttcgctg aaaatgaagt 840taaaccttta gcaacagaac ttgatgaaga agaaagattt ccttatgaaa cagtggaaaa 900aatggcaaaa gcaggaatga tgggtatacc atatccaaaa gaatatggtg gagaaggtgg 960agacactgta ggatatataa tggcagttga agaattgtct agagtttgtg gtactacagg 1020agttatatta tcagctcata catctcttgg ctcatggcct atatatcaat atggtaatga 1080agaacaaaaa caaaaattct taagaccact agcaagtgga gaaaaattag gagcatttgg 1140tcttactgag cctaatgctg gtacagatgc gtctggccaa caaacaactg ctgttttaga 1200cggggatgaa tacatactta atggctcaaa aatatttata acaaacgcaa tagctggtga 1260catatatgta gtaatggcaa tgactgataa atctaagggg aacaaaggaa tatcagcatt 1320tatagttgaa aaaggaactc ctgggtttag ctttggagtt aaagaaaaga aaatgggtat 1380aagaggttca gctacgagtg aattaatatt tgaggattgc agaataccta aagaaaattt 1440acttggaaaa gaaggtcaag gatttaagat agcaatgtct actcttgatg gtggtagaat 1500tggtatagct gcacaagctt taggtttagc acaaggtgct cttgatgaaa ctgttaaata 1560tgtaaaagaa agagtacaat ttggtagacc attatcaaaa ttccaaaata cacaattcca 1620attagctgat atggaagtta aggtacaagc ggctagacac cttgtatatc aagcagctat 1680aaataaagac ttaggaaaac cttatggagt agaagcagca atggcaaaat tatttgcagc 1740tgaaacagct atggaagtta ctacaaaagc tgtacaactt catggaggat atggatacac 1800tcgtgactat ccagtagaaa gaatgatgag agatgctaag ataactgaaa tatatgaagg 1860aactagtgaa gttcaaagaa tggttatttc aggaaaacta ttaaaatagt aagaaggaga 1920tatacatatg gaggaaggat ttatgaatat agtcgtttgt ataaaacaag ttccagatac 1980aacagaagtt aaactagatc ctaatacagg tactttaatt agagatggag taccaagtat 2040aataaaccct gatgataaag caggtttaga agaagctata aaattaaaag aagaaatggg 2100tgctcatgta actgttataa caatgggacc tcctcaagca gatatggctt taaaagaagc 2160tttagcaatg ggtgcagata gaggtatatt attaacagat agagcatttg cgggtgctga 2220tacttgggca acttcatcag cattagcagg agcattaaaa aatatagatt ttgatattat 2280aatagctgga agacaggcga tagatggaga tactgcacaa gttggacctc aaatagctga 2340acatttaaat cttccatcaa taacatatgc tgaagaaata aaaactgaag gtgaatatgt 2400attagtaaaa agacaatttg aagattgttg ccatgactta aaagttaaaa tgccatgcct 2460tataacaact cttaaagata tgaacacacc aagatacatg aaagttggaa gaatatatga 2520tgctttcgaa aatgatgtag tagaaacatg gactgtaaaa gatatagaag ttgacccttc 2580taatttaggt cttaaaggtt ctccaactag tgtatttaaa tcatttacaa aatcagttaa 2640accagctggt acaatataca atgaagatgc gaaaacatca gctggaatta tcatagataa 2700attaaaagag aagtatatca tataataaga aggagatata catatgggta acgttttagt 2760agtaatagaa caaagagaaa atgtaattca aactgtttct ttagaattac taggaaaggc 2820tacagaaata gcaaaagatt atgatacaaa agtttctgca ttacttttag gtagtaaggt 2880agaaggttta atagatacat tagcacacta tggtgcagat gaggtaatag tagtagatga 2940tgaagcttta gcagtgtata caactgaacc atatacaaaa gcagcttatg aagcaataaa 3000agcagctgac cctatagttg tattatttgg tgcaacttca ataggtagag atttagcgcc 3060tagagtttct gctagaatac atacaggtct tactgctgac tgtacaggtc ttgcagtagc 3120tgaagataca aaattattat taatgacaag acctgccttt ggtggaaata taatggcaac 3180aatagtttgt aaagatttca gacctcaaat gtctacagtt agaccagggg ttatgaagaa 3240aaatgaacct gatgaaacta aagaagctgt aattaaccgt ttcaaggtag aatttaatga 3300tgctgataaa ttagttcaag ttgtacaagt aataaaagaa gctaaaaaac aagttaaaat 3360agaagatgct aagatattag tttctgctgg acgtggaatg ggtggaaaag aaaacttaga 3420catactttat gaattagctg aaattatagg tggagaagtt tctggttctc gtgccactat 3480agatgcaggt tggttagata aagcaagaca agttggtcaa actggtaaaa ctgtaagacc 3540agacctttat atagcatgtg gtatatctgg agcaatacaa catatagctg gtatggaaga 3600tgctgagttt atagttgcta taaataaaaa tccagaagct ccaatattta aatatgctga 3660tgttggtata gttggagatg ttcataaagt gcttccagaa cttatcagtc agttaagtgt 3720tgcaaaagaa aaaggtgaag ttttagctaa ctaataagaa ggagatatac atatgagaga 3780agtagtaatt gccagtgcag ctagaacagc agtaggaagt tttggaggag catttaaatc 3840agtttcagcg gtagagttag gggtaacagc agctaaagaa gctataaaaa gagctaacat 3900aactccagat atgatagatg aatctctttt agggggagta cttacagcag gtcttggaca 3960aaatatagca agacaaatag cattaggagc aggaatacca gtagaaaaac cagctatgac 4020tataaatata gtttgtggtt ctggattaag atctgtttca atggcatctc aacttatagc 4080attaggtgat gctgatataa tgttagttgg tggagctgaa aacatgagta tgtctcctta 4140tttagtacca agtgcgagat atggtgcaag aatgggtgat gctgcttttg ttgattcaat 4200gataaaagat ggattatcag acatatttaa taactatcac atgggtatta ctgctgaaaa 4260catagcagag caatggaata taactagaga agaacaagat gaattagctc ttgcaagtca 4320aaataaagct gaaaaagctc aagctgaagg aaaatttgat gaagaaatag ttcctgttgt 4380tataaaagga agaaaaggtg acactgtagt agataaagat gaatatatta agcctggcac 4440tacaatggag aaacttgcta agttaagacc tgcatttaaa aaagatggaa cagttactgc 4500tggtaatgca tcaggaataa atgatggtgc tgctatgtta gtagtaatgg ctaaagaaaa 4560agctgaagaa ctaggaatag agcctcttgc aactatagtt tcttatggaa cagctggtgt 4620tgaccctaaa ataatgggat atggaccagt tccagcaact aaaaaagctt tagaagctgc 4680taatatgact attgaagata tagatttagt tgaagctaat gaggcatttg ctgcccaatc 4740tgtagctgta ataagagact taaatataga tatgaataaa gttaatgtta atggtggagc 4800aatagctata ggacatccaa taggatgctc aggagcaaga atacttacta cacttttata 4860tgaaatgaag agaagagatg ctaaaactgg tcttgctaca ctttgtatag gcggtggaat 4920gggaactact ttaatagtta agagatagta agaaggagat atacatatga aattagctgt

4980aataggtagt ggaactatgg gaagtggtat tgtacaaact tttgcaagtt gtggacatga 5040tgtatgttta aagagtagaa ctcaaggtgc tatagataaa tgtttagctt tattagataa 5100aaatttaact aagttagtta ctaagggaaa aatggatgaa gctacaaaag cagaaatatt 5160aagtcatgtt agttcaacta ctaattatga agatttaaaa gatatggatt taataataga 5220agcatctgta gaagacatga atataaagaa agatgttttc aagttactag atgaattatg 5280taaagaagat actatcttgg caacaaatac ttcatcatta tctataacag aaatagcttc 5340ttctactaag cgcccagata aagttatagg aatgcatttc tttaatccag ttcctatgat 5400gaaattagtt gaagttataa gtggtcagtt aacatcaaaa gttacttttg atacagtatt 5460tgaattatct aagagtatca ataaagtacc agtagatgta tctgaatctc ctggatttgt 5520agtaaataga atacttatac ctatgataaa tgaagctgtt ggtatatatg cagatggtgt 5580tgcaagtaaa gaagaaatag atgaagctat gaaattagga gcaaaccatc caatgggacc 5640actagcatta ggtgatttaa tcggattaga tgttgtttta gctataatga acgttttata 5700tactgaattt ggagatacta aatatagacc tcatccactt ttagctaaaa tggttagagc 5760taatcaatta ggaagaaaaa ctaagatagg attctatgat tataataaat aataagaagg 5820agatatacat atgagtacaa gtgatgttaa agtttatgag aatgtagctg ttgaagtaga 5880tggaaatata tgtacagtga aaatgaatag acctaaagcc cttaatgcaa taaattcaaa 5940gactttagaa gaactttatg aagtatttgt agatattaat aatgatgaaa ctattgatgt 6000tgtaatattg acaggggaag gaaaggcatt tgtagctgga gcagatattg catacatgaa 6060agatttagat gctgtagctg ctaaagattt tagtatctta ggagcaaaag cttttggaga 6120aatagaaaat agtaaaaaag tagtgatagc tgctgtaaac ggatttgctt taggtggagg 6180atgtgaactt gcaatggcat gtgatataag aattgcatct gctaaagcta aatttggtca 6240gccagaagta actcttggaa taactccagg atatggagga actcaaaggc ttacaagatt 6300ggttggaatg gcaaaagcaa aagaattaat ctttacaggt caagttataa aagctgatga 6360agctgaaaaa atagggctag taaatagagt cgttgagcca gacattttaa tagaagaagt 6420tgagaaatta gctaagataa tagctaaaaa tgctcagctt gcagttagat actctaaaga 6480agcaatacaa cttggtgctc aaactgatat aaatactgga atagatatag aatctaattt 6540atttggtctt tgtttttcaa ctaaagacca aaaagaagga atgtcagctt tcgttgaaaa 6600gagagaagct aactttataa aagggtaata agaaggagat atacatatga gaagttttga 6660agaagtaatt aagtttgcaa aagaaagagg acctaaaact atatcagtag catgttgcca 6720agataaagaa gttttaatgg cagttgaaat ggctagaaaa gaaaaaatag caaatgccat 6780tttagtagga gatatagaaa agactaaaga aattgcaaaa agcatagaca tggatatcga 6840aaattatgaa ctgatagata taaaagattt agcagaagca tctctaaaat ctgttgaatt 6900agtttcacaa ggaaaagccg acatggtaat gaaaggctta gtagacacat caataatact 6960aaaagcagtt ttaaataaag aagtaggtct tagaactgga aatgtattaa gtcacgtagc 7020agtatttgat gtagagggat atgatagatt atttttcgta actgacgcag ctatgaactt 7080agctcctgat acaaatacta aaaagcaaat catagaaaat gcttgcacag tagcacattc 7140attagatata agtgaaccaa aagttgctgc aatatgcgca aaagaaaaag taaatccaaa 7200aatgaaagat acagttgaag ctaaagaact agaagaaatg tatgaaagag gagaaatcaa 7260aggttgtatg gttggtgggc cttttgcaat tgataatgca gtatctttag aagcagctaa 7320acataaaggt ataaatcatc ctgtagcagg acgagctgat atattattag ccccagatat 7380tgaaggtggt aacatattat ataaagcttt ggtattcttc tcaaaatcaa aaaatgcagg 7440agttatagtt ggggctaaag caccaataat attaacttct agagcagaca gtgaagaaac 7500taaactaaac tcaatagctt taggtgtttt aatggcagca aaggcataat aagaaggaga 7560tatacatatg agcaaaatat ttaaaatctt aacaataaat cctggttcga catcaactaa 7620aatagctgta tttgataatg aggatttagt atttgaaaaa actttaagac attcttcaga 7680agaaatagga aaatatgaga aggtgtctga ccaatttgaa tttcgtaaac aagtaataga 7740agaagctcta aaagaaggtg gagtaaaaac atctgaatta gatgctgtag taggtagagg 7800aggacttctt aaacctataa aaggtggtac ttattcagta agtgctgcta tgattgaaga 7860tttaaaagtg ggagttttag gagaacacgc ttcaaaccta ggtggaataa tagcaaaaca 7920aataggtgaa gaagtaaatg ttccttcata catagtagac cctgttgttg tagatgaatt 7980agaagatgtt gctagaattt ctggtatgcc tgaaataagt agagcaagtg tagtacatgc 8040tttaaatcaa aaggcaatag caagaagata tgctagagaa ataaacaaga aatatgaaga 8100tataaatctt atagttgcac acatgggtgg aggagtttct gttggagctc ataaaaatgg 8160taaaatagta gatgttgcaa acgcattaga tggagaagga cctttctctc cagaaagaag 8220tggtggacta ccagtaggtg cattagtaaa aatgtgcttt agtggaaaat atactcaaga 8280tgaaattaaa aagaaaataa aaggtaatgg cggactagtt gcatacttaa acactaatga 8340tgctagagaa gttgaagaaa gaattgaagc tggtgatgaa aaagctaaat tagtatatga 8400agctatggca tatcaaatct ctaaagaaat aggagctagt gctgcagttc ttaagggaga 8460tgtaaaagca atattattaa ctggtggaat cgcatattca aaaatgttta cagaaatgat 8520tgcagataga gttaaattta tagcagatgt aaaagtttat ccaggtgaag atgaaatgat 8580tgcattagct caaggtggac ttagagtttt aactggtgaa gaagaggctc aagtttatga 8640taactaataa 8650136862DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 13gtaaaacgac ggccagtgaa ttcgttaaga cccactttca catttaagtt gtttttctaa 60tccgcatatg atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa 120taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc 180ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac 240agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa 300ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat gtacttttgc 360tccatcgcga tgacttagta aagcacatct aaaactttta gcgttattac gtaaaaaatc 420ttgccagctt tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat 480ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc 540tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag 600cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatca ttaattccta 660atttttgttg acactctatc attgatagag ttattttacc actccctatc agtgatagag 720aaaagtgaac tctagaaata attttgttta actttaagaa ggagatatac atatgatcgt 780aaaacctatg gtacgcaaca atatctgcct gaacgcccat cctcagggct gcaagaaggg 840agtggaagat cagattgaat ataccaagaa acgcattacc gcagaagtca aagctggcgc 900aaaagctcca aaaaacgttc tggtgcttgg ctgctcaaat ggttacggcc tggcgagccg 960cattactgct gcgttcggat acggggctgc gaccatcggc gtgtcctttg aaaaagcggg 1020ttcagaaacc aaatatggta caccgggatg gtacaataat ttggcatttg atgaagcggc 1080aaaacgcgag ggtctttata gcgtgacgat cgacggcgat gcgttttcag acgagatcaa 1140ggcccaggta attgaggaag ccaaaaaaaa aggtatcaaa tttgatctga tcgtatacag 1200cttggccagc ccagtacgta ctgatcctga tacaggtatc atgcacaaaa gcgttttgaa 1260accctttgga aaaacgttca caggcaaaac agtagatccg tttactggcg agctgaagga 1320aatctccgcg gaaccagcaa atgacgagga agcagccgcc actgttaaag ttatgggggg 1380tgaagattgg gaacgttgga ttaagcagct gtcgaaggaa ggcctcttag aagaaggctg 1440tattaccttg gcctatagtt atattggccc tgaagctacc caagctttgt accgtaaagg 1500cacaatcggc aaggccaaag aacacctgga ggccacagca caccgtctca acaaagagaa 1560cccgtcaatc cgtgccttcg tgagcgtgaa taaaggcctg gtaacccgcg caagcgccgt 1620aatcccggta atccctctgt atctcgccag cttgttcaaa gtaatgaaag agaagggcaa 1680tcatgaaggt tgtattgaac agatcacgcg tctgtacgcc gagcgcctgt accgtaaaga 1740tggtacaatt ccagttgatg aggaaaatcg cattcgcatt gatgattggg agttagaaga 1800agacgtccag aaagcggtat ccgcgttgat ggagaaagtc acgggtgaaa acgcagaatc 1860tctcactgac ttagcggggt accgccatga tttcttagct agtaacggct ttgatgtaga 1920aggtattaat tatgaagcgg aagttgaacg cttcgaccgt atctgataag aaggagatat 1980acatatgaga gaagtagtaa ttgccagtgc agctagaaca gcagtaggaa gttttggagg 2040agcatttaaa tcagtttcag cggtagagtt aggggtaaca gcagctaaag aagctataaa 2100aagagctaac ataactccag atatgataga tgaatctctt ttagggggag tacttacagc 2160aggtcttgga caaaatatag caagacaaat agcattagga gcaggaatac cagtagaaaa 2220accagctatg actataaata tagtttgtgg ttctggatta agatctgttt caatggcatc 2280tcaacttata gcattaggtg atgctgatat aatgttagtt ggtggagctg aaaacatgag 2340tatgtctcct tatttagtac caagtgcgag atatggtgca agaatgggtg atgctgcttt 2400tgttgattca atgataaaag atggattatc agacatattt aataactatc acatgggtat 2460tactgctgaa aacatagcag agcaatggaa tataactaga gaagaacaag atgaattagc 2520tcttgcaagt caaaataaag ctgaaaaagc tcaagctgaa ggaaaatttg atgaagaaat 2580agttcctgtt gttataaaag gaagaaaagg tgacactgta gtagataaag atgaatatat 2640taagcctggc actacaatgg agaaacttgc taagttaaga cctgcattta aaaaagatgg 2700aacagttact gctggtaatg catcaggaat aaatgatggt gctgctatgt tagtagtaat 2760ggctaaagaa aaagctgaag aactaggaat agagcctctt gcaactatag tttcttatgg 2820aacagctggt gttgacccta aaataatggg atatggacca gttccagcaa ctaaaaaagc 2880tttagaagct gctaatatga ctattgaaga tatagattta gttgaagcta atgaggcatt 2940tgctgcccaa tctgtagctg taataagaga cttaaatata gatatgaata aagttaatgt 3000taatggtgga gcaatagcta taggacatcc aataggatgc tcaggagcaa gaatacttac 3060tacactttta tatgaaatga agagaagaga tgctaaaact ggtcttgcta cactttgtat 3120aggcggtgga atgggaacta ctttaatagt taagagatag taagaaggag atatacatat 3180gaaattagct gtaataggta gtggaactat gggaagtggt attgtacaaa cttttgcaag 3240ttgtggacat gatgtatgtt taaagagtag aactcaaggt gctatagata aatgtttagc 3300tttattagat aaaaatttaa ctaagttagt tactaaggga aaaatggatg aagctacaaa 3360agcagaaata ttaagtcatg ttagttcaac tactaattat gaagatttaa aagatatgga 3420tttaataata gaagcatctg tagaagacat gaatataaag aaagatgttt tcaagttact 3480agatgaatta tgtaaagaag atactatctt ggcaacaaat acttcatcat tatctataac 3540agaaatagct tcttctacta agcgcccaga taaagttata ggaatgcatt tctttaatcc 3600agttcctatg atgaaattag ttgaagttat aagtggtcag ttaacatcaa aagttacttt 3660tgatacagta tttgaattat ctaagagtat caataaagta ccagtagatg tatctgaatc 3720tcctggattt gtagtaaata gaatacttat acctatgata aatgaagctg ttggtatata 3780tgcagatggt gttgcaagta aagaagaaat agatgaagct atgaaattag gagcaaacca 3840tccaatggga ccactagcat taggtgattt aatcggatta gatgttgttt tagctataat 3900gaacgtttta tatactgaat ttggagatac taaatataga cctcatccac ttttagctaa 3960aatggttaga gctaatcaat taggaagaaa aactaagata ggattctatg attataataa 4020ataataagaa ggagatatac atatgagtac aagtgatgtt aaagtttatg agaatgtagc 4080tgttgaagta gatggaaata tatgtacagt gaaaatgaat agacctaaag cccttaatgc 4140aataaattca aagactttag aagaacttta tgaagtattt gtagatatta ataatgatga 4200aactattgat gttgtaatat tgacagggga aggaaaggca tttgtagctg gagcagatat 4260tgcatacatg aaagatttag atgctgtagc tgctaaagat tttagtatct taggagcaaa 4320agcttttgga gaaatagaaa atagtaaaaa agtagtgata gctgctgtaa acggatttgc 4380tttaggtgga ggatgtgaac ttgcaatggc atgtgatata agaattgcat ctgctaaagc 4440taaatttggt cagccagaag taactcttgg aataactcca ggatatggag gaactcaaag 4500gcttacaaga ttggttggaa tggcaaaagc aaaagaatta atctttacag gtcaagttat 4560aaaagctgat gaagctgaaa aaatagggct agtaaataga gtcgttgagc cagacatttt 4620aatagaagaa gttgagaaat tagctaagat aatagctaaa aatgctcagc ttgcagttag 4680atactctaaa gaagcaatac aacttggtgc tcaaactgat ataaatactg gaatagatat 4740agaatctaat ttatttggtc tttgtttttc aactaaagac caaaaagaag gaatgtcagc 4800tttcgttgaa aagagagaag ctaactttat aaaagggtaa taagaaggag atatacatat 4860gagaagtttt gaagaagtaa ttaagtttgc aaaagaaaga ggacctaaaa ctatatcagt 4920agcatgttgc caagataaag aagttttaat ggcagttgaa atggctagaa aagaaaaaat 4980agcaaatgcc attttagtag gagatataga aaagactaaa gaaattgcaa aaagcataga 5040catggatatc gaaaattatg aactgataga tataaaagat ttagcagaag catctctaaa 5100atctgttgaa ttagtttcac aaggaaaagc cgacatggta atgaaaggct tagtagacac 5160atcaataata ctaaaagcag ttttaaataa agaagtaggt cttagaactg gaaatgtatt 5220aagtcacgta gcagtatttg atgtagaggg atatgataga ttatttttcg taactgacgc 5280agctatgaac ttagctcctg atacaaatac taaaaagcaa atcatagaaa atgcttgcac 5340agtagcacat tcattagata taagtgaacc aaaagttgct gcaatatgcg caaaagaaaa 5400agtaaatcca aaaatgaaag atacagttga agctaaagaa ctagaagaaa tgtatgaaag 5460aggagaaatc aaaggttgta tggttggtgg gccttttgca attgataatg cagtatcttt 5520agaagcagct aaacataaag gtataaatca tcctgtagca ggacgagctg atatattatt 5580agccccagat attgaaggtg gtaacatatt atataaagct ttggtattct tctcaaaatc 5640aaaaaatgca ggagttatag ttggggctaa agcaccaata atattaactt ctagagcaga 5700cagtgaagaa actaaactaa actcaatagc tttaggtgtt ttaatggcag caaaggcata 5760ataagaagga gatatacata tgagcaaaat atttaaaatc ttaacaataa atcctggttc 5820gacatcaact aaaatagctg tatttgataa tgaggattta gtatttgaaa aaactttaag 5880acattcttca gaagaaatag gaaaatatga gaaggtgtct gaccaatttg aatttcgtaa 5940acaagtaata gaagaagctc taaaagaagg tggagtaaaa acatctgaat tagatgctgt 6000agtaggtaga ggaggacttc ttaaacctat aaaaggtggt acttattcag taagtgctgc 6060tatgattgaa gatttaaaag tgggagtttt aggagaacac gcttcaaacc taggtggaat 6120aatagcaaaa caaataggtg aagaagtaaa tgttccttca tacatagtag accctgttgt 6180tgtagatgaa ttagaagatg ttgctagaat ttctggtatg cctgaaataa gtagagcaag 6240tgtagtacat gctttaaatc aaaaggcaat agcaagaaga tatgctagag aaataaacaa 6300gaaatatgaa gatataaatc ttatagttgc acacatgggt ggaggagttt ctgttggagc 6360tcataaaaat ggtaaaatag tagatgttgc aaacgcatta gatggagaag gacctttctc 6420tccagaaaga agtggtggac taccagtagg tgcattagta aaaatgtgct ttagtggaaa 6480atatactcaa gatgaaatta aaaagaaaat aaaaggtaat ggcggactag ttgcatactt 6540aaacactaat gatgctagag aagttgaaga aagaattgaa gctggtgatg aaaaagctaa 6600attagtatat gaagctatgg catatcaaat ctctaaagaa ataggagcta gtgctgcagt 6660tcttaaggga gatgtaaaag caatattatt aactggtgga atcgcatatt caaaaatgtt 6720tacagaaatg attgcagata gagttaaatt tatagcagat gtaaaagttt atccaggtga 6780agatgaaatg attgcattag ctcaaggtgg acttagagtt ttaactggtg aagaagaggc 6840tcaagtttat gataactaat aa 6862141420DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 14ttattatcgc accgcaatcg ggattttcga ttcataaagc aggtcgtagg tcggcttgtt 60gagcaggtct tgcagcgtga aaccgtccag atacgtgaaa aacgacttca ttgcaccgcc 120gagtatgccc gtcagccggc aggacggcgt aatcaggcat tcgttgttcg ggcccataca 180ctcgaccagc tgcatcggtt cgaggtggcg gacgaccgcg ccgatattga tgcgttcggg 240cggcgcggcc agcctcagcc cgccgccttt cccgcgtacg ctgtgcaaga acccgccttt 300gaccagcgcg gtaaccactt tcatcaaatg gcttttggaa atgccgtagg tcgaggcgat 360ggtggcgata ttgaccagcg cgtcgtcgtt gacggcggtg tagatgagga cgcgcagccc 420gtagtcggta tgttgggtca gatacataca acctccttag tacatgcaaa attatttcta 480gagcaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagttgagtt 540gaggaattat aacaggaaga aatattcctc atacgcttgt aattcctcta tggttgttga 600caattaatca tcggctcgta taatgtataa cattcatatt ttgtgaattt taaactctag 660aaataatttt gtttaacttt aagaaggaga tatacatatg gctagcaaag gcgaagaatt 720gttcacgggc gttgttccta ttttggttga attggatggc gatgttaatg gccataaatt 780cagcgttagc ggcgaaggcg aaggcgatgc tacgtatggc aaattgacgt tgaaattcat 840ttgtacgacg ggcaaattgc ctgttccttg gcctacgttg gttacgacgt tcagctatgg 900cgttcaatgt ttcagccgtt atcctgatca tatgaaacgt catgatttct tcaaaagcgc 960tatgcctgaa ggctatgttc aagaacgtac gattagcttc aaagatgatg gcaattataa 1020aacgcgtgct gaagttaaat tcgaaggcga tacgttggtt aatcgtattg aattgaaagg 1080cattgatttc aaagaagatg gcaatatttt gggccataaa ttggaatata attataatag 1140ccataatgtt tatattacgg ctgataaaca aaaaaatggc attaaagcta atttcaaaat 1200tcgtcataat attgaagatg gcagcgttca attggctgat cattatcaac aaaatacgcc 1260tattggcgat ggccctgttt tgttgcctga taatcattat ttgagcacgc aaagcgcttt 1320gagcaaagat cctaatgaaa aacgtgatca tatggttttg ttggaattcg ttacggctgc 1380tggcattacg catggcatgg atgaattgta taaataataa 142015967DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 15caaatatcac ataatcttaa catatcaata aacacagtaa agtttcatgt gaaaaacatc 60aaacataaaa tacaagctcg gaatacgaat cacgctatac acattgctaa caggaatgag 120attatctaaa tgaggattga tatattaatt ggacatacta gtttttttca tcaaaccagt 180agagataact tccttcacta tctcaatgag gaagaaataa aacgctatga tcagtttcat 240tttgtgagtg ataaagaact ctatatttta agccgtatcc tgctcaaaac agcactaaaa 300agatatcaac ctgatgtctc attacaatca tggcaattta gtacgtgcaa atatggcaaa 360ccatttatag tttttcctca gttggcaaaa aagatttttt ttaacctttc ccatactata 420gatacagtag ccgttgctat tagttctcac tgcgagcttg gtgtcgatat tgaacaaata 480agagatttag acaactctta tctgaatatc agtcagcatt tttttactcc acaggaagct 540actaacatag tttcacttcc tcgttatgaa ggtcaattac ttttttggaa aatgtggacg 600ctcaaagaag cttacatcaa atatcgaggt aaaggcctat ctttaggact ggattgtatt 660gaatttcatt taacaaataa aaaactaact tcaaaatata gaggttcacc tgtttatttc 720tctcaatgga aaatatgtaa ctcatttctc gcattagcct ctccactcat cacccctaaa 780ataactattg agctatttcc tatgcagtcc caactttatc accacgacta tcagctaatt 840cattcgtcaa atgggcagaa ttgaatcgcc acggataatc tagacacttc tgagccgtcg 900ataatattga ttttcatatt ccgtcggtgg tgtaagtatc ccgcataatc gtgccattca 960catttag 96716424DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 16ggatgggggg aaacatggat aagttcaaag aaaaaaaccc gttatctctg cgtgaaagac 60aagtattgcg catgctggca caaggtgatg agtactctca aatatcacat aatcttaaca 120tatcaataaa cacagtaaag tttcatgtga aaaacatcaa acataaaata caagctcgga 180atacgaatca cgctatacac attgctaaca ggaatgagat tatctaaatg aggattgatg 240tgtaggctgg agctgcttcg aagttcctat actttctaga gaataggaac ttcggaatag 300gaacttcgga ataggaacta aggaggatat tcatatgtcg tcaaatgggc agaattgaat 360cgccacggat aatctagaca cttctgagcc gtcgataata ttgattttca tattccgtcg 420gtgg 424

Claims

1-31. (canceled)

32. A genetically engineered bacterium comprising: a) at least one non-native copy of a first gene that encodes a transcription factor protein that is regulated by a reactive nitrogen species (RNS), wherein the first gene is operatively linked to a promoter; and b) at least one of: i. a second gene encoding a non-native, anti-inflammation molecule; ii. a second gene encoding a non-native gut barrier function enhancer molecule; iii. a gene cassette encoding a biosynthetic pathway, wherein a final product of the biosynthetic pathway is an anti-inflammation molecule; iv. a gene cassette encoding a biosynthetic pathway, wherein a final product of the biosynthetic pathway is a gut barrier function enhancer molecule, wherein the second gene or gene cassette in b) is expressed under the control of a tunable regulatory region heterologous to the gene or gene cassette, wherein induction of the tunable regulatory region is directly or indirectly controlled by the transcription factor.

33. The bacterium of claim 32, wherein at least one of the one or more non-native copies of the gene that encodes the transcription factor is located on a plasmid in the bacterium.

34. The bacterium of claim 32, wherein at least one of the one or more non-native copies of the gene that encodes the transcription factor is located on a chromosome in the bacterium.

35. The bacterium of claim 32, wherein the promoter that controls expression of at least one of the one or more non-native copies of the gene that encodes the transcription factor is an inducible promoter.

36. The bacterium of claim 32, wherein the gene encoding the anti-inflammation molecule, the gut barrier enhancer molecule, or the gene cassette encoding the biosynthetic pathway is located on a plasmid in the bacterium.

37. The bacterium of claim 32, wherein the gene encoding the anti-inflammation molecule, the gut barrier enhancer molecule, or the gene cassette encoding the biosynthetic pathway is located on a chromosome in the bacterium.

38. The bacterium of claim 32, wherein the gene that encodes the transcription factor protein is nitric oxide sensing repressor NsrR.

39. The bacterium of claim 32, wherein the tunable regulatory region that controls expression of the anti-inflammation molecule, the gut barrier enhancer molecule, or the biosynthetic pathway is selected from a native or a modified functional form of a regulatory region from any one of nitric oxide reductase (norB), aniA, nsrR, hmpA, ytfE, ygbA, hcp, hcr, nrfA, and alternative oxidase (aox).

40. The bacterium of claim 32, wherein the molecule of b) is selected from propionate, butyrate, acetate, interleukin 10 (IL-10), interleukin 27 (IL-27), transforming growth factor .beta.2 (TGF- 2), transforming growth factor .beta.1 (TGF- 1), glucagon-like peptide (GLP-2), N-acylphosphatidylethanolamines (NAPEs), elafin, trefoil factor, and single-chain variable fragment (scFv), antisense RNA, short interfering RNA (siRNA), or short hairpin RNA (shRNA) directed against a pro-inflammatory molecule.

41. The bacterium of claim 32, wherein the bacterium is a non-pathogenic bacterium.

42. The bacterium of claim 41, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.

43. The bacterium of claim 42, wherein the bacterium is Escherichia coli strain Nissle.

44. The bacterium of claim 32, wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.

45. A pharmaceutically acceptable composition comprising the bacterium of claim 32; and a pharmaceutically acceptable carrier.

46. The composition of claim 45 formulated for oral or rectal administration.

47. A method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, the composition of claim 45.

48. 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 claim 45.

49. The method of claim 47, 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.

50. The method of claim 49, wherein the autoimmune disorder is selected from the group consisting of type 1 diabetes, asthma, multiple sclerosis, Crohn's disease, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, celiac disease, and ankylosing spondylitis.

51. The method of claim 48, wherein the disease or disorder is selected from an inflammatory bowel disease, and a diarrheal disease.