WO2017075485A1

WO2017075485A1 - Bacteria engineered to treat disorders in which trimethylamine (tma) is detrimental

Info

Publication number: WO2017075485A1
Application number: PCT/US2016/059518
Authority: WO
Inventors: Vincent M. Isabella; Paul F. Miller; Dean Falb; Jonathan W. Kotula
Original assignee: Synlogic, Inc.
Priority date: 2015-10-30
Filing date: 2016-10-28
Publication date: 2017-05-04

Abstract

The present invention provides recombinant bacterial cells comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s). The recombinant bacterial cells optionally comprise gene sequence encoding one or more TMA and/or TMAO transporter(s). The invention further provides pharmaceutical compositions comprising the recombinant bacteria, and methods for treating disorders in which trimethylamine (TMA) and/or TMAO is detrimental, such as cardiovascular disease, kidney disease, and/or trimethylaminuria, using the pharmaceutical compositions of the invention.

Description

BACTERIA ENGINEERED TO TREAT

DISORDERS IN WHICH TRIMETHYLAMINE (TMA) IS DETRIMENTAL

Related Applications

[01] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 62/248,615, filed October 30, 2015, U.S. Provisional Patent

Application No. 62/249,620, filed November 2, 2015, U.S. Provisional Patent Application No. 62/255,757, filed November 16, 2015, and PCT Application No. PCT/US2016/032565, filed May 13, 2016, the contents of which are hereby incorporated by reference herein in their entirety.

Background

[02] In mammals, dietary choline and 1-carnitine, which are present in egg yolk, beans, high- fat dairy products, and meat, are metabolized into trimethylamine (TMA) by the colonic microflora and absorbed by passive diffusion across cell membranes (Koeth et al., 2013, Nature Med., 19:576-585; Mackay et al, 2011, Clin. Biochem. Rev., 32(l):33-43). Additionally, gamma-butyrobetaine, a new intermediate of 1-carnitine metabolism, has been shown to be converted into TMA in the lower gut (Koeth et al, 2014, Cell Metab.,

20(5):799-812). TMA then enters the enterohepatic circulation and is removed by the liver. In normal liver cells, flavin-containing monooxygenases (FMOs) rapidly convert TMA into TMAO, which is water soluble and is secreted mostly in the urine (Tang et al., 2015, Circ. Res., 116(3):448-455; Tang ei a/., 2014, J. Am. Coll. Cardiol, 64(18): 1908-1914).

[03] Subjects with trimethylaminuria have an autosomal recessive mutation in the FM03 gene, which encodes an enzyme that breaks down nitrogen-containing compounds from the diet, including trimethylamine. Excess TMA builds up in subjects having trimethylaminuria and is released via sweat, urine, and breath, causing a characteristic odor of a "fishy smell." This characteristic "fishy smell" can interfere with many aspects of daily life and may affect the relationships, social life, and career of a subject having trimethylaminuria.

[04] Moreover, subjects suffering from trimethylaminuria have also been shown to have increased risk of cardiovascular disease (Zschocke et al., 1999, Lancet, 354(9181):834- 835; Mackay et al., 2011, Clin. Biochem. Rev., 32(l):33-43). Recent studies have shown that TMA and TMAO play a role in cholesterol metabolism, vascular inflammation, and the formation of unstable plaques in arterial walls, and that TMA and TMAO contribute to the development of cardiovascular disease, atherosclerosis, heart failure, and kidney disease (Gregory et al, 2015, J. Biol. Chem., 290(9):5647-5660; Moraes et al, 2015, J. Ren. Nutr., 25(6):459-465; Tang et al, 2014, Circulation Research, 116(3):448). Indeed, oral dietary supplementation with TMAO or the TMA-containing precursors, choline or carnitine, in mice has been shown to enhance atherosclerotic plaque in a mouse model of disease (Gregory et al, 2015; J. Biol. Chem., 290(9):5647-5660). TMAO, is also elevated in chronic kidney diseases (CKD) and is associated with both higher risk of progressive renal fibrosis and functional impairment, and poorer long-term survival (Tang et al., Gut Microbiota-Dependent Trimethylamine N-oxide (TMAO) Pathway Contributes to Both Development of Renal Insufficiency and Mortality Risk in Chronic Kidney Disease, Circ Res. 2015 Jan 30; 116(3): 448-455).

[05] There are no current treatments for trimethylaminuria other than avoidance of foods containing TMA and its precursors {e.g., choline, lecithin, and TMAO), such as fish, eggs, beans, soy products, and red meat. Thus, a need exists for treatments which address the build-up of TMA and TMAO in subjects in order to treat and/or prevent cardiovascular disease, kidney disease, and trimethylaminuria.

Summary

[06] The present disclosure provides engineered microorganisms, e.g., bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating disorders in which trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) is detrimental. In some embodiments, the bacteria cell comprises endogenous gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s) and can naturally metabolize TMA and/or TMAO, and have been engineered to increase the expression and/or activity of the endogenous genes. In some embodiments, a microorganism, e.g., bacterial cell, has been engineered to comprise heterologous gene sequence encoding one or more trimethylamine catabolism enzyme(s). In some embodiments, the microorganism, e.g., bacterial cell, engineered to comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s) is capable of processing and reducing levels of trimethylamine and/or

trimethylamine N-oxide. In some embodiments, the microorganism, e.g., bacterial cell, engineered to comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s) is capable of processing and reducing levels of trimethylamine and/or

trimethylamine N-oxide in low-oxygen environments, e.g., such those environments found in the gut. Thus, the engineered microorganisms, e.g., bacterial cells, and pharmaceutical compositions comprising the microorganisms, e.g., bacterial cells, may be used to convert excess trimethylamine and/or trimethylamine N-oxide into non-toxic molecules in order to treat and/or prevent conditions associated with disorders in which trimethylamine is detrimental, such as cardiovascular disease, kidney disease, and/or trimethylaminuria.

[07] In one aspect, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s). In some

embodiments, the one or more trimethylamine catabolism enzyme is a trimethylamine dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a dimethylamine dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a γ-glutamylmethylamine synthetase. In some embodiments, the one or more trimethylamine catabolism enzyme is a N-methylglutamate synthase. In some embodiments, the one or more trimethylamine catabolism enzyme is a N-methylglutamate dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a formaldehyde dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a formate dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a methanol dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a methylamine-glutamate N- methyltransferase.

[08] In some embodiments, the present disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) selected from a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, and a γ- glutamylmethylamine synthetase. In some embodiments, the bacterial cell further comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) selected from an N-methylglutamate synthase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, and a formate dehydrogenase. In some embodiments, the bacterial cell further comprises gene sequence encoding a methanol dehydrogenase. In some

embodiments, the bacterial cell comprises gene sequence encoding a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, a γ-glutamylmethylamine synthetase, an N- methylglutamate synthase, an N-methylglutamate dehydrogenase, a formaldehyde

dehydrogenase, and a formate dehydrogenase. In some embodiments, the bacterial cell further comprises gene sequence encoding a methanol dehydrogenase.

[09] In some embodiments, the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) selected from a trimethylamine

dehydrogenase, a dimethylamine dehydrogenase, a methylamine-glutamate N- methyltransferase, and an N-methylglutamate dehydrogenase. In some embodiments, the bacterial cell further comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) selected from a formaldehyde dehydrogenase and a formate dehydrogenase. In some embodiments, the bacterial cell further comprises gene sequence encoding a methanol dehydrogenase. In some embodiments, the bacterial cell comprises gene sequence encoding a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, a methylamine-glutamate N-methyltransferase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, and a formate dehydrogenase. In some embodiments, the bacterial cell further comprises gene sequence encoding a methanol dehydrogenase.

[010] In some embodiments, the gene sequence encoding one or more

trimethylamine catabolism enzyme(s) is a heterologous gene. In some embodiments, the gene sequence encoding one or more trimethylamine catabolism enzyme(s) is located on a plasmid in the bacterial cell. In other embodiments, the gene sequence encoding one or more trimethylamine catabolism enzyme(s) is located on a chromosome in the bacterial cell.

[011] In one aspect, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter. In some embodiments, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by

environmental conditions. In some embodiments, the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by low oxygen or anaerobic conditions, such as conditions found in the gut, e.g., a mammalian gut. In specific embodiments, the inducible promoter is directly or indirectly induced by environmental conditions specific to the small intestine of a mammal. In some embodiments, the first inducible promoter is an FNR responsive promoter. In some embodiments, the FNR responsive promoter is selected from a promoter comprising SEQ ID NO:4, a promoter comprising SEQ ID NO:5, a promoter comprising SEQ ID NO:6, a promoter comprising SEQ ID NO:7, a promoter comprising SEQ ID NO:8, a promoter comprising SEQ ID NO: 19, a promoter comprising SEQ ID NO:20, a promoter comprising SEQ ID NO:21, a promoter comprising SEQ ID NO:22, a promoter comprising SEQ ID NO:23, a promoter comprising SEQ ID NO:24, a promoter comprising SEQ ID NO:25, a promoter comprising SEQ ID NO:26, a promoter comprising SEQ ID NO:27, a promoter comprising SEQ ID NO:28, a promoter comprising SEQ ID NO:29, and a promoter comprising SEQ ID NO:30. [012] In some embodiments, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by inflammation or inflammatory conditions or an inflammatory response, such as inflammatory conditions or an inflammatory response that may be present in the gut. In some embodiments, the bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) is operably linked to an inducible promoter that is directly or indirectly induced by the presence or absence of reactive oxygen species (ROS). In other embodiments, the bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) is operably linked to an inducible promoter that is directly or indirectly induced by the presence or absence of reactive nitrogen species (RNS). In some embodiments, the bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) is operably linked to an inducible promoter that is directly or indirectly induced by a biological molecule(s) that is involved in the inflammatory response, for example, a molecule present in an inflammatory disorder of the gut. In other embodiments, the exogenous environmental condition(s) or signal(s) that induces the promoter exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides, e.g. , the gut or small intestine. In some embodiments, the bacterial cell comprising gene sequence encoding one or more

trimethylamine catabolism enzyme(s) is operably linked to an inducible promoter that is induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. In some embodiments, the exogenous environmental condition(s) or signal(s) are artificially created, for example, by the creation or removal of a biological condition(s) and/or the administration or removal of a biological molecule(s). In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response).

[013] In one aspect, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) and gene sequence encoding one or more TMA and/or TMAO transporter(s) that imports trimethylamine and/or trimethylamine-N-oxide into the bacterial cell. In some embodiments, the bacterial cell comprising gene sequence encoding one or more TMA and/or TMAO transporter(s) is operably linked to an inducible promoter. In some embodiments, the bacterial cell comprising gene sequence encoding one or more TMA and/or TMAO transporter(s) is operably linked to an inducible promoter that is directly or indirectly induced by

environmental conditions, e.g., conditions found in the gut, including, for example, low oxygen or anaerobic conditions, inflammation, inflammatory conditions or an inflammatory response, a metabolite that may or may not be naturally present in the gut, such as any of the inducible promoters disclosed herein. In some embodiments, the bacterial cell comprising gene sequence encoding one or more TMA and/or TMAO transporter(s) is operably linked to a constitutive promoter. In some embodiments, the gene sequence encoding one or more TMA and/or TMAO transporter(s) is a heterologous gene. In some embodiments, the gene sequence encoding one or more TMA and/or TMAO transporter(s) is located on a plasmid in the bacterial cell. In other embodiments, the gene sequence encoding one or more TMA and/or TMAO transporter(s) is located on a chromosome in the bacterial cell.

[014] In some embodiments, the bacterial cell is an auxotroph in a gene that is complemented when the bacterial cell is present in a mammalian gut. In some embodiments, the mammalian gut is a human gut. In some embodiments, the bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine bio synthetic pathway.

[015] In some embodiments, the bacterial cell additionally comprises gene sequence encoding at least one substance that is toxic to the bacterial cell. In some embodiments, the gene sequence encoding at least one substance that is toxic is operably linked to a second inducible promoter. In some embodiments, the expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the at least one

trimethylamine catabolism enzyme. In some embodiments, the gene sequence encoding at least one substance that is toxic is operably linked to a second inducible promoter and the expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the at least one trimethylamine catabolism enzyme. Thus, in some embodiments, the invention provides a bacterial cell comprising gene sequence encoding at least one trimethylamine catabolism enzyme operably linked to a first inducible promoter and a heterologous gene encoding a substance that is toxic to the bacterial cell that is operably linked to a second inducible promoter, wherein expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the at least one trimethylamine catabolism enzyme. In some embodiments, the second inducible promoter is directly or indirectly induced by environmental conditions. In some embodiments, the second inducible promoter is directly or indirectly induced by environmental conditions found in the gut, e.g., a mammalian gut. In some embodiments, the second inducible promoter is directly or indirectly induced by environmental conditions specific to the small intestine of a mammal. In some embodiments, the second inducible promoter is directly or indirectly induced by low-oxygen or anaerobic conditions. In some embodiments, the second inducible promoter is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut. In some embodiments, the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In other embodiments, the first inducible promoter and the second inducible promoter are different promoters. In other embodiments, the gene sequence encoding the at least one trimethylamine catabolism enzyme is operably linked to a constitutive promoter. In some embodiments, the heterologous gene encoding a substance that is toxic to the bacterial cell is operably linked to a consitutive promoter.

[016] In some embodiments, the bacterial cell is a recombinant bacterial cell. In some embodiments, the bacterial cell is a probiotic bacterial cell. In some embodiments, the bacterial cell is a recombinant probiotic bacterial cell. In some embodiments, the bacterial cell is a member of a genus selected from the group consisting of Bacteroides,

Bifidobacterium, Clostridium, Escherichia, Lactobacillus and Lactococcus. In some embodiments, the bacterial cell is of the genus Escherichia. In some embodiments, the bacterial cell is of the species Escherichia coli strain Nissle.

[017] In one aspect, the disclosure provides a composition comprising a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a composition comprising a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter, and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a composition comprising a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by an environmental condition, and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a composition comprising a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by environmental conditions found in the gut, e.g. , a mammalian gut, and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a composition comprising a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by environmental condition found in the small intestine, and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a composition comprising a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to an inducible promoter that is directly or indirectly induced by low oxygen or anaerobic conditions, and a pharmaceutically acceptable carrier.

[018] In some embodiments of the pharmaceutical composition, the one or more trimethylamine catabolism enzyme(s) is selected from a trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamine synthetase, N-methylglutamate synthase, N-methylglutamate dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, methanol dehydrogenase and methylamine-glutamate N-methyltransferase. In some embodiments, the one or more trimethylamine catabolism(s) is a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, and a γ-glutamylmethylamine synthetase, and optionally an N-methylglutamate synthase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, and a formate dehydrogenase, and further optionally a methanol dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme is a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, a γ- glutamylmethylamine synthetase, an N-methylglutamate synthase, an N-methylglutamate dehydrogenase, and optionally a formaldehyde dehydrogenase and a formate dehydrogenase, and further optionally a methanol dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme(s) is a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, a methylamine-glutamate N-methyltransferase, and an N-methylglutamate dehydrogenase and optionally a formaldehyde dehydrogenase and a formate dehydrogenase, and further optionally a methanol dehydrogenase. In some embodiments, the one or more trimethylamine catabolism enzyme(s) is a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, a methylamine-glutamate N-methyltransferase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, and a formate dehydrogenase, and optionally a methanol dehydrogenase.

[019] In any of these embodiments of the pharmaceutical composition, the one or more gene(s) encoding at least one trimethylamine catabolism enzyme is a heterologous gene. In any one of these embodiments, the one or more gene(s) encoding at least one trimethylamine catabolism enzyme is located on a plasmid in the bacterial cell or is located on a chromosome in the bacterial cell. In any of these embodiments, the bacterial cell is an auxotroph in a gene that is complemented when the bacterial cell is present in a mammalian gut. In any of these embodiments, the bacterial cell further comprises a heterologous gene encoding a substance that is toxic to the bacterial cell that is operably linked to directly or indirectly inducible promoter, wherein expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the at least one

trimethylamine catabolism enzyme.

[020] In one aspect, the disclosure provides a method for treating a disease in which TMA and/or TMAO is detrimental in a subject, the method comprising administering a bacterial cell disclosed herein, or a pharmaceutical composition comprising a bacterial cell disclosed herein (such as any of the pharmaceutical compositions disclosed above or elsewhere herein) to the subject, wherein the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s). In some embodiments, the invention provides a method for treating a disease in which TMA and/or TMAO is detrimental in a subject, the method comprising administering a bacterial cell disclosed herein, or a pharmaceutical composition comprising a bacterial cell disclosed herein (such as any of the pharmaceutical compositions disclosed above or elsewhere herein), to the subject, wherein the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) which is expressed in response to an exogenous environmental condition in the subject, thereby treating the disease in which TMA and/or TMAO is detrimental in the subject.

[021] In another aspect, the disclosure provides a method for treating a disease in which trimethylamine and/or trimethylamine-N-oxide is detrimental in a subject, the method comprising administering a pharmaceutical composition of the disclosure to the subject, thereby treating the disease in which trimethylamine and/or trimethylamine-N-oxide is detrimental in the subject.

[022] In one aspect, the disclosure provides a method for preventing a disease in which TMA and/or TMAO is detrimental in a subject, the method comprising administering a bacterial cell disclosed herein, or a pharmaceutical composition comprising a bacterial cell disclosed herein (such as any of the pharmaceutical compositions disclosed above or elsewhere herein) to the subject, wherein the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s). In some embodiments, the invention provides a method for preventing a disease in which TMA and/or TMAO is detrimental in a subject, the method comprising administering a bacterial cell disclosed herein, or a pharmaceutical composition comprising a bacterial cell disclosed herein (such as any of the pharmaceutical compositions disclosed above or elsewhere herein), to the subject, wherein the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) which is expressed in response to an exogenous environmental condition in the subject, thereby preventing the disease in which TMA and/or TMAO is detrimental in the subject.

[023] In another aspect, the disclosure provides a method for decreasing a level of trimethylamine in the gut of a subject, the method comprising administering a pharmaceutical composition of the disclosure to the subject, thereby decreasing the level of trimethylamine and/or trimethylamine-N-oxide in the gut of the subject.

[024] In another aspect, the disclosure provides a method for decreasing a level of trimethylamine and/or trimethylamine-N-oxide in the sweat of a subject, the method comprising administering a pharmaceutical composition of the disclosure to the subject, thereby decreasing the level of trimethylamine in the sweat of the subject.

[025] In another aspect, the disclosure provides a method for decreasing a level of trimethylamine and/or trimethylamine-N-oxide in the urine of a subject, the method comprising administering a pharmaceutical composition of the disclosure to the subject, thereby decreasing the level of trimethylamine in the urine of the subject.

[026] In another aspect, the disclosure provides a method for decreasing a level of trimethylamine and/or trimethylamine N-oxide (TMAO) in the blood or plasma of a subject, the method comprising administering a pharmaceutical composition of the invention disclosure to the subject, thereby decreasing the level of TMAO in the blood or plasma of the subject.

[027] In one embodiment, the level of trimethylamine and/or trimethylamine-N- oxide is decreased in plasma of the subject after administering the pharmaceutical

composition to the subject. In one embodiment, the level of trimethylamine and/or trimethylamine N-oxide in plasma of the subject is decreased at least two-fold after administering the pharmaceutical composition to the subject. In another embodiment, the level of trimethylamine and/or trimethylamine-N-oxide in plasma of the subject is decreased at least 3-, 4-, or 5-fold after administering the pharmaceutical composition to the subject.

[028] In one embodiment, the level of trimethylamine and/or trimethylamine N- oxide is reduced in urine of the subject after administering the pharmaceutical composition to the subject. In another embodiment, the level of trimethylamine and/or trimethylamine N- oxide is decreased at least two-fold in urine of the subject after administering the pharmaceutical composition to the subject. In another embodiment, the level of

trimethylamine and/or trimethylamine N-oxide is decreased at least 3-, 4-, or 5-fold in urine of the subject after administering the pharmaceutical composition to the subject.

[029] In one embodiment, the pharmaceutical composition is administered orally. In one embodiment, the subject is a human subject.

[030] In one embodiment, the method further comprises isolating a plasma sample from the subject or a urine sample from the subject after administering the pharmaceutical composition to the subject, and determining the level of trimethylamine and/or

trimethylamine N-oxide in the plasma sample from the subject or the urine sample from the subject.

[031] In one embodiment, the method further comprises comparing the level of trimethylamine and/or trimethylamine N-oxide in the plasma sample from the subject or the urine sample from the subject to a control level of trimethylamine and/or trimethylamine N- oxide. In one embodiment, the control level of trimethylamine and/or trimethylamine N- oxide is the level of trimethylamine and/or trimethylamine N-oxide in the plasma of the subject or in the urine of the subject before administration of the pharmaceutical composition. In another embodiment, the control level is a daily urinary excretion of a ratio of greater than 92% of TMAO/(TMAO+TMA).

[032] In one embodiment, the subject is considered treated when the subject exhibits a daily urinary excretion of a ratio of greater than 92% of TMAO/(TMAO+TMA).

[033] In one embodiment, the subject has a disorder in which trimethylamine and/or trimethylamine N-oxide is detrimental. In one embodiment, the disorder in which

trimethylamine is detrimental is trimethylaminuria. In another embodiment, the disorder in which trimethylamine and/or trimethylamine N-oxide is detrimental is a cardiovascular disease. In one embodiment, the cardiovascular disease is atherosclerosis. In another embodiment, the disorder in which trimethylamine and/or trimethylamine N-oxide is detrimental is kidney disease.

[034] In one embodiment, the subject is fed a meal within one hour of administering the pharmaceutical composition. In another embodiment, the subject is fed a meal concurrently with administering the pharmaceutical composition. Brief Description of the Drawings

[035] Figs. 1A and IB depict schemes of circuits for trimethylamine (TMA) catabolism for use in the recombinant bacteria. To briefly summarize, the following enzymatic activities are engineered in the recombinant bacteria in order to get low oxygen or anaerobic degradation of TMA to carbon dioxide and NADH: trimethylamine

dehydrogenase, dimethylamine dehydrogenase, gamma-glutamylmethylamine synthetase, N- methylglutamate dehydrogenase, methanol dehydrogenase, formaldehyde dehydrogenase*, and formate dehydrogenase* (Fig. 1A). In another embodiment, the following enzymatic activities are engineered in the recombinant bacterial eel in order to get low oxygen or anaerobic degradation of TMA to gamma-glutamylmethylamide: trimethylamine

dehydrogenase, dimethylamine dehydrogenase, gamma-glutamylmethylamine synthetase (Fig. 1A). In another embodiment, the following enzymatic activities are engineered in the recombinant bacterial of the invention in order to get anaerobic degradation of TMA to carbon dioxide and NADH: trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine-glutamate N-methyltransferase, N-methylglutamate dehydrogenase,

formaldehyde dehydrogenase*, and formade dehydrogenase* (Fig. IB). Note that enzymes indicated with a * are naturally present in E. coli and certain other bacteria.

[036] Fig. 2 depicts the state of one non-limiting embodiment of the TMA enzyme construct under inducing conditions. Specifically, Fig. 2 depicts up-regulated TMA enzyme production under low oxygen or anaerobic conditions due to FNR dimerization and induction of FNR responsive promoter-mediated expression of the genes in a recombinant bacterial cell. Each arrow adjacent to one or a cluster of rectangles depicts the promoter responsible for driving transcription, in the direction of the arrow, of such gene(s). Arrows above each rectangle depict the expression product of each gene.

[037] Fig. 3A depicts a schematic of the structures of choline, carnitine,

trimethylamine, and trimethylamine n-oxide. Fig. 3B depicts a schematic of

proatherosclerotic mechanisms of trimethylamine (TMA), e.g., as described in Koeth et al., Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes

atherosclerosis; Nat Med. 2013 May; 19(5): 576-585, the contents of which is herein incorporated by reference in its entirety. Gut microbiota produce TMA from carnitine and/or choline, which is converted into trimethylamine-N-oxide (TMAO) by flavin monooxygenases (FMO) in the liver. TMAO acts through at least four proatherosclerotic mechanisms on cholesterol metabolism: 1. Reduction of reverse cholesterol transport. Reverse cholesterol transport is a multi-step process resulting in the net movement of cholesterol from peripheral tissues back to the liver via the plasma. Cholesterol from non-hepatic peripheral tissues is transferred to HDL by the ABCA1 (ATP-binding cassette transporter). 2. Cell surface expression of two proatherogenic scavenger receptors, cluster of differentiation 36 (CD36) and scavenger receptor A (SRA) and foam cell formation. 3. Reduction in bile acid pool. TMAO promotes reduction in expression of Cyp7al, the major bile acid synthetic enzyme and rate limiting step in the catabolism of cholesterol. TMAO reduces bile acid synthesis and bile acid secretion. 4. TMAO promotes changes in microbiota in the intestine. TMAO also reduces expression of both intestinal cholesterol transporters, Nieman-Pick Cl-like 1

(NpclLl), which transports cholesterol inot enterocyte from the gut lumen, and Abcg5/8, which trasnports cholesterol out of enterocytes into the gut lumen. In some embodiments, the genetically engineered bacteria are useful for the treatment, management and/or prevention of cardiovascular disease. In some embodiments, the genetically engineered bacteria take up and catabolize TMA and/or TMAO.

[038] Fig. 4 depicts a schematic illustrating the role of TMAO in chronic kidney disease, e.g., as described in Tang et al., Gut Microbiota-Dependent Trimethylamine N-oxide (TMAO) Pathway Contributes to Both Development of Renal Insufficiency and Mortality Risk in Chronic Kidney Disease, Circ Res. 2015 Jan 30; 116(3): 448-455, the contents of which is herein incorporated by reference in its entirety. Gut microbiota produce TMA from carnitine and/or choline, which is converted into trimethylamine-N-oxide (TMAO) by flavin monooxygenases (FMO) in the liver. TMAO is transported to the kidneys, where it is cleared. TMAO is elevated in subjects with impaired renal function, and promotes renal injury. Levels of the early kidney injury marker KIM-1 are increased, and phosphorylation of Smad3, an important regulator of renal fibrosis is enhanced. Plasma levels of cystatin C, a sensitive indicator of renal functional impairment, are increased. In some embodiments, the genetically engineered bacteria are useful for the treatment, management and/or prevention of chronic kidney disease. In some embodiments, the genetically engineered bacteria take up and catabolize TMA and/or TMAO.

[039] Fig. 5 depicts a map of integration sites within the E. coli Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites. [040] Fig. 6 depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent.

[041] Fig. 7 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (Mo As).

[042] Fig. 8 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[043] Fig. 9 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N- terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta- domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.

[044] Fig. 10 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP- binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[045] Fig. 11 depicts a schematic of the outer and inner membranes of a gram- negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the extracellular space, e.g., therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., Ipp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.

[046] Fig. 12 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g., a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons).

[047] Fig. 13A, Fig. 13B, and Fig. 13C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide described herein, is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (Fig. 13A and Fig. 13B) or a tet-inducible promoter (Fig. 13C). In alternate embodiments, an inducible promoter, for example, an oxygen level-dependent promoter (e.g., FNR-inducible promoter), promoter induced by a disease specific molecule (e.g., kidney disease or arthero sclerosis specific molecules), promoter induced by

inflammation or an inflammatory response (RNS, ROS promoters), and/or promoter induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose is used. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion oifliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct.

[048] Fig. 14A and Fig. 14B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments the genetically engineered bacteria comprise deletions in one or more of Ipp, pal, tolA, and/or nlpl. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a tet promoter (Fig. 14A) or an inducible promoter, such as an oxygen level-dependent promoter (e.g., FNR- inducible promoter, Fig. 14B), promoter induced by a disease specific molecule (e.g. , kidney disease or arthero sclerosis specific molecules), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and/or promoter induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g. , arabinose.

[049] Fig. 15A, Fig. 15B, and Fig. 15C depict schematics of other non-limiting embodiments of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (ParaBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti- toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. Fig. 15A also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell. Fig. 15B depicts a non- limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit. Fig. 15C depicts another non- limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.

[050] Fig. 16 depicts one non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated

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

[051] Fig. 17 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti- toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.

[052] Fig. 18 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the

recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death.

[053] Fig. 19 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[054] Fig. 20 depicts a one non- limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived antitoxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce a equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti-toxin decays completely, and the cell dies.

[055] Fig. 21 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al, "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synthetic Biology (2015) 4: 307-316.

[056] Fig. 22 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown in Tables 2 and 3 (Pfnrl-5). Different FNR- responsive promoters were used to create a library of anaerobic-inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+0₂) or anaerobic conditions (-0₂). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays.

[057] Fig. 23A, Fig. 23B and Fig. 23C depict schematic representations of the lacZ gene under the control of an exemplary FNR promoter

and corresponding graphical data. Fig. 23A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (Pfnrs)- LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. Fig. 23B depicts FNR promoter activity as a function of β- galactosidase activity in SYN340. SYN340, an engineered bacterial strain harboring a low- copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr under anaerobic conditions. Fig. 23C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

[058] Fig. 24A, Fig. 24B, Fig. 24C, and Fig. 24D depict bar graphs, schematic, and dot blot, respectively, showing the structure or activity of reporter constructs. Fig. 24A and Fig. 24B depict bar graphs of reporter constructs activity. Fig. 24A depicts a graph of an ATC-inducible reporter construct expression and Fig. 24B depicts a graph of a nitric oxide- inducible reporter construct expression. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible P_tet-GFP reporter construct or the nitric oxide inducible P_nsrR-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units. Fig. 24C depicts a schematic of the constructs. Fig. 24D depicts a dot blot of bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR- inducible promoter. DSS-treated mice serve as exemplary models for HE. As in HE subjects, the guts of mice are damaged by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice.

[059] Fig. 25A depicts a graph showing bacterial cell growth of a Nissle thyA auxotroph strain (thyA knock-out) in various concentrations of thymidine. A

chloramphenicol-resistant Nissle thyA auxotroph strain was grown overnight in LB + lOmM thymidine at 37°C. The next day, cells were diluted 1: 100 in 1 mL LB + lOmM thymidine, and incubated at 37°C for 4 hours. The cells were then diluted 1: 100 in 1 mL LB + varying concentrations of thymidine in triplicate in a 96-well plate. The plate is incubated at 37°C with shaking, and the OD600 is measured every 5 minutes for 720 minutes. This data shows that Nissle thyA auxotroph does not grow in environments lacking thymidine.

[060] Fig. 25B depicts a bar graph of Nissle residence in vivo of wildtype Nissle versus Nissle thyA auxotroph (thyA knock-out). Streptomycin- resistant Nissle (wildtype or thyA auxotroph) was administered to mice via oral gavage without antibiotic pre- treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. Each bar represents the number of Nissle recovered from the fecal samples each day for 7 consecutive days. There were no bacteria recovered in fecal samples from mice gavaged with Nissle thyA auxotroph bacteria after day 3. This data shows that the Nissle thyA auxotroph does not persist in vivo in mice.

[061] Fig. 26 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

[062] Fig. 27 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 10⁹ CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating.

[063] Fig. 28A and Fig. 28B depict a schematic diagrams of a wild-type clbA construct (Fig. 29A) and a schematic diagram of a clbA knockout construct (Fig. 28B).

[064] Fig. 29 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animla disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen.

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

Detailed Description

[066] The present disclosure provides engineered microorganisms, e.g. , bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating disorders in which trimethylamine (TMA) and/or trimethylamine-N-oxide (TMAO) is detrimental. In some embodiments, a microorganism, e.g. , bacterial cell, has been engineered to comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s). In some embodiments, the microorganism, e.g. , bacterial cell, engineered to comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s) is capable of processing and reducing levels of trimethylamine and/or trimethylamine N-oxide. In some embodiments, the microorganism, e.g. , bacterial cell, engineered to comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s) is capable of processing and reducing levels of trimethylamine and/or trimethylamine N-oxide in low-oxygen environments, e.g., such those environments found in the gut. Thus, the engineered microorganisms, e.g. , bacterial cells, and pharmaceutical compositions comprising the microorganisms, e.g. , bacterial cells, may be used to convert excess trimethylamine and/or trimethylamine N-oxide into non-toxic molecules in order to treat and/or prevent conditions associated with disorders in which trimethylamine is detrimental, such as cardiovascular disease, kidney disease, and/or trimethylaminuria.

[067] In one aspect, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s). In one aspect, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to a first inducible promoter. In some embodiments, the disclosure provides a bacterial cell comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to a first inducible promoter that is directly or indirectly induced by environmental conditions, such as by low oxygen or anaerobic conditions, such as conditions found in the gut or small intestine of a mammal. In some embodiments, a bacterial cell of the invention has been genetically engineered to comprise at least one heterologous gene encoding at least one trimethylamine catabolism enzyme and is capable of processing and reducing levels of trimethylamine and/or trimethylamine-N-oxide, in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells of the disclosure may be used to convert excess TMA and/or TMAO into non-toxic molecules in order to treat and/or prevent conditions associated with disorders in which trimethylamine and/or trimethylamine-N-oxide is detrimental, such as cardiovascular disease, kidney disease, and/or trimethylaminuria. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding enzymes which allow the depletion of TMA and/or TMAO leading to improved cardiorenal health (reduced atherosclerosis and kidney dysfunction) and mortality.

[068] Specifically, the engineered bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, at least one trimethylamine catabolism enzyme. In some embodiments, the engineered bacteria additionally comprise optional circuitry to ensure the safety and non-colonization of the subject that is administered the engineered bacteria, such as auxotrophies, kill switches, etc. These engineered bacteria are safe and well tolerated and augment the innate activities of the subject's microbiome to achieve a therapeutic effect.

[069] 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.

[070] As used herein, "microorganism" refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, yeast, parasites, fungi, certain algae, and protozoa. In some aspects, the microorganism is engineered ("engineered

microorganism") to produce one or more therpauetic molecules, e.g., lysosomal enzyme(s). In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus. In certain embodiments, the engineered microorganism is an engineered yeast.

[071] As used herein, the term "recombinant microorganism" refers to a

microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state. Thus, a "recombinant bacterial cell" or

"recombinant bacteria" refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

[072] A "programmed microorganism" or "engineered microorganism" refers to a microorganism, e.g. , bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state to perform a specific function, e.g., to process and reduce levels of trimethylamine and/or trimethylamine-N-oxide , for example, in low-oxygen environments. In certain embodiments, the programmed or engineered microorganism has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered microorganism may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.

[073] A "programmed bacterial cell" or "engineered bacterial cell" is a bacterial cell that has been genetically modified from its native state. In certain embodiments, the programmed or engineered bacterial cell has been modified from its native state to perform a specific function, for example, to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose, e.g. , to process and reduce levels of trimethylamine and/or trimethylamine-N-oxide , for example, in low-oxygen environments. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed. For instance, an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

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

[075] The term "gene" is meant to encompass full-length gene sequences (e.g. , as found in nature and/or a gene sequence encoding a full-length polypeptide or protein) and is also meant to include partial gene sequences (e.g. , a fragment of the gene sequence found in nature and/or a gene sequence encoding a protion or fragment of a polypeptide or protein). The term "gene" is meant to encompass modified gene sequences (e.g., modified as compared to the sequence found in nature). Thus, the term "gene" is not limited to the natural or full-length gene sequence found in nature.

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

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

"endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, the term "transgene" refers to a gene that has been introduced into the host organism, e.g. , host bacterial cell, genome.

[078] As used herein, a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a microorganism, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria or virus, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria or virus of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g. , Purcell et ah , 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, "non- native" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the genetically engineered microorganism of the disclosure comprises a gene that is operably linked to a promoter that is not associated with said gene in nature. For example, in some embodiments, the genetically engineered bacteria disclosed herein comprise a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g. , an FNR responsive promoter (or other promoter disclosed herein) operably linked to a gene encoding at least one trimethylamine catabolism enzyme. In some embodiments, the genetically engineered virus of the disclosure comprises a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., a promoter operably linked to a gene encoding a trimethylamine catabolism enzyme.

[079] As used herein, the term "coding region" refers to a nucleotide sequence that codes for a specific amino acid sequence. The term "regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, signal sequences, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter or other promoter disclosed herein. [080] As used herein, "stably maintained" or "stable" bacterium is used to refer to a bacterial host cell carrying non- native genetic material, e.g. , a gene encoding at least one trimethylamine catabolism enzyme, which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium is capable of survival and/or growth in vitro, e.g. , in medium, and/or in vivo, e.g. , in the gut. For example, the stable bacterium may be a genetically engineered bacterium comprising a gene encoding a at least one trimethylamine catabolism enzyme catabolism enzyme, in which the plasmid or chromosome carrying the gene is stably maintained in the bacterium, such that the trimethylamine catabolism enzyme can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.

[081] As used herein, a "gene cassette" or "operon" encoding a trimethylamine catabolism pathway or a trimethylamine and/or trimethylamine-N-oxide catabolism pathway refers to the two or more genes that are required to catabolize TMA and/or TMAO into nontoxic molecules. 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. Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.

[082] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding a trimethylamine catabolism enzyme, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the trimethylamine catabolism enzyme. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be "directly linked" to a regulatory sequence in a manner which allows expression of the gene. In another embodiment, a gene may be "indirectly linked" to a regulatory sequence in a manner which allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes.

A regulatory region or sequence is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[083] A "promoter" as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5' of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue- specific manner, in response to different environmental or physiological conditions, or in response to specific compounds.

Prokaryotic promoters are typically classified into two classes: inducible and constitutive. A "constitutive promoter" refers to a promoter that allows for continual transcription of the coding sequence or gene under its control.

[084] "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli σ promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli σ³² promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ 70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis σ^Α romoter {e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), Pi_iaG (BBa_K823000), Pi_epA

(BBa_K823002), P_veg (BBa_K823003)), a constitutive Bacillus subtilis σ promoter {e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter {e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter {e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_Kl 13010; BBa_Kl 13011 ; BBa_Kl 13012; BBa_R0085; BBa_R0180; BBa_R0181 ; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter {e.g., SP6 promoter (BBa_J64998)).

[085] An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. An "inducible promoter" refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A "directly inducible promoter" refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An "indirectly inducible promoter" refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by "inducible promoter." Exemplary inducible promoters described herein include oxygen level-dependent promoters {e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present {e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. Examples of inducible promoters include, but are not limited to, an FNR responsive promoter, a Parac promoter, a ParaBAD promoter, and a PietR promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.

[086] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide. [087] As used herein, the term "plasmid" or "vector" refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell' s genome.

Plasmids are usually circular and capable of autonomous replication. Plasmids may be low- copy, medium-copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid may comprise a nucleic acid sequence encoding one or more heterologous gene(s) or gene cassette(s), e.g., encoding at least one trimethylamine catabolism enzyme.

[088] As used herein, the term "transform" or "transformation" refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically- stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.

[089] The term "genetic modification," as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, base substitution, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising at least one trimethylamine catabolism enzyme operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation.

[090] As used herein, the term "genetic mutation" refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example,

substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term "genetic mutation" is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene's polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.

[091] The term "genetic modification that increases import of trimethylamine into the bacterial cell" and the term "genetic modification that increases import of trimethylamine and/or trimethylamine-N-oxide into the bacterial cell" refer to a genetic modification that increases the uptake rate or increases the uptake quantity of trimethylamine and/or trimethylamine-N-oxide into the cytosol or periplasm of the bacterial cell, as compared to the uptake rate or uptake quantity of the trimethylamine and/or trimethylamine-N-oxide into the cytosol or periplasm of a bacterial cell not having said modification, e.g., a wild-type bacterial cell. In some embodiments, an engineered bacterial cell having a genetic

modification that increases import of trimethylamine and/or trimethylamine-N-oxide into the bacterial cell refers to a bacterial cell comprising heterologous gene sequence (native or non- native) encoding one or more importer(s) (transporter(s)) of trimethylamine and/or trimethylamine-N-oxide. In some embodiments, the engineered bacteria comprising genetic modification that increases import of trimethylamine and/or trimethylamine-N-oxide into the bacterial cell comprise gene sequence(s) encoding a trimethylamine and/or trimethylamine- N-oxide transporter. The transporter can be any transporter that assists or allows import of trimethylamine and/or trimethylamine-N-oxide into the cell. In some embodiments, the engineered bacteria comprise more than one copy of gene sequence encoding a

trimethylamine and/or trimethylamine-N-oxide transporter. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding more than one trimethylamine and/or trimethylamine-N-oxide transporter, e.g. , two or more different trimethylamine and/or trimethylamine-N-oxide transporters. In some embodiments, the transporter is able to transport both trimethylamine and trimethylamine-N-oxide. In some embodiments, the transporter is able to transport trimethylamine or trimethylamine-N-oxide. In some embodiments, an engineered bacterial cell having a genetic modification that increases import of trimethylamine and/or trimethylamine-N-oxide into the bacterial cell comprises a genetic mutation in a native gene. In another embodiment, an engineered bacterial cell having a genetic modification that increases import of a trimethylamine and/or trimethylamine-N- oxide into the bacterial cell comprises a genetic mutation in a native promoter, which increases or activates transcription of the gene which increases import of trimethylamine and/or trimethylamine-N-oxide. In another embodiment, an engineered bacterial cell having a genetic modification that increases import of trimethylamine and/or trimethylamine-N- oxide into the bacterial cell comprises a genetic mutation leading to overexpression of an activator of an importer (transporter) of trimethylamine and/or trimethylamine-N-oxide. In another embodiment, an engineered bacterial cell having a genetic modification that increases import of trimethylamine and/or trimethylamine-N-oxide into the bacterial cell comprises a genetic mutation in an importer (transporter) of trimethylamine and/or trimethylamine-N- oxide that leads to increased activity of the transporter. In another embodiment, an engineered bacterial cell having a genetic modification that increases import of

trimethylamine and/or trimethylamine-N-oxide into the bacterial cell comprises a genetic mutation which increases or activates translation of the gene encoding the transporter (importer).

[092] As used herein, the term "transporter" is meant to refer to a mechanism, e.g. , protein, proteins, or protein complex, for importing a molecule, e.g. , amino acid, peptide (di- peptide, tri-peptide, polypeptide, etc), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.

[093] As used herein, the term "trimethylamine transporter" or "TMA transporter" "trimethylamine and/or trimethylamine-N-oxide transporter" or "TMA and/or TMAO transporter" refers to a polypeptide which functions to transport trimethylamine and/or trimethylamine-N-oxide into the bacterial cell.

[094] The term "trimethylamine" or "TMA," as used herein, refers to a compound of the formula N(CH₃)₃. Trimethylamine is converted to trimethylamine N-oxide (TMAO) in the liver of subjects. The term "trimethylamine N-oxide" or "TMAO" refers to a compound having the formula (CH₃)₃NO. TMA and its co-metabolite TMAO are associated with cardiovascular disease (CVD), and TMA formation requires gut bacteria, which have been shown to TMA from L-carnitine and choline (see e.g., Zhang and Davies, Microbial metabolism of dietary components to bioactive metabolites: opportunities for new therapeutic interventions; Genome Med. 2016; 8: 46). After formation and absorption in the colon, TMA passes into the portal circulation, and into the liver, where it is oxidized to TMAO by flavin- containing mono-oxygenase 3 (FM03). TMAO levels predict risk for atherosclerosis, and are elevated in patients with chronic kidney disease (CKD) and obesity. TMAO directly induces CVD, as administration of TMAO itself or of sufficient choline or L-carnitine to raise TMAO levels can all increase atherosclerosis in Apoe-/- mice (Koeth et al.). Accumulation of TMAO in the kidney aloters osmotic balance and elevated TMAO levels associate in animal models with markers of renal damage such fibrosis and dysfunction. Thus, interventions to reduce CVD, chronic kidney disease and/or obesity focus on reducing TMAO levels. In some embodiments, the geneticially engineered bacteria comprise one or more gene sequence(s) encoding enzymes which reduce TMA and/or TMAO levels.

[095] The term "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.

[096] The term "non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii,

Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al, 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity. [097] As used herein, the term "treat" and its cognates refer to an amelioration of a disease, 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, 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. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease.

[098] Those in need of treatment may include individuals already having a particular medical disease, as well as those at risk of having, or who may ultimately acquire the disease. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely receptiveness to treatment of a subject having the disease. Diseases associated with the catabolism of trimethylamine and/or trimethylamine-N-oxide, e.g., cardiovascular disease, kidney disease, or trimethylaminuria, may be caused by inborn genetic mutations for which there are no known cures. Diseases can also be secondary to other conditions, e.g., an intestinal disorder or a bacterial infection. Treating diseases in which trimethylamine and/or trimethylamine-N-oxide is detrimental may encompass reducing normal levels of

trimethylamine and/or trimethylamine-N-oxide, reducing excess levels of trimethylamine and/or trimethylamine-N-oxide, or eliminating trimethylamine and/or trimethylamine-N- oxide, and does not necessarily encompass the elimination of the underlying disease.

[099] As used herein, the term "catabolism" refers to the processing, breakdown and/or degradation of a complex molecule, such as trimethylamine and/or trimethylamine-N- oxide, into compounds that are non-toxic or which can be utilized by the bacterial cell. In one embodiment, the term "trimethylamine catabolism" refers to the processing, breakdown, and/or degradation of trimethylamine into γ-glutamylmethylamide (see, for example, Fig. IB). In another embodiment, the term "trimethylamine catabolism" refers to the processing, breakdown, and/or degradation of trimethylamine into carbon dioxide and NADH (see, for example, Fig. 1A). In some embodiments, "trimethylamine catabolism" or "TMA

catabolism" or "trimethylamine and/or trimethylamine-N-oxide catabolism" or "TMA and/or TMAO catabolism" includes the catabolism of TMAO, e.g. , the conversion of TMAO to TMA by a TMAO reductase.

[0100] In one embodiment, "abnormal catabolism" refers to any condition(s), disorder(s), disease(s), predisposition(s), and/or genetic mutations(s) that result in increased levels of trimethylamine (TMA) and/or trimethylamine-N-oxide (TMAO). In one embodiment, abnormal catabolism refers to a daily urinary excretion of a ratio of less than 85% of TMAO/(TMAO+TMA) (see, for example, Wevers et al, 2008, Laboratory Guide to the Methods in Biochemical Genetics, Springer- Ver lag; New York, 2008, p. 781-792; and Mackay et al, 2011, Clin. Biochem. Rev., 32(l):33-43). Subjects with normal

trimethylamine catabolism typically have a ratio of greater than 92% of

TMAO/(TMAO+TMA). In one embodiment, "abnormal catabolism" refers to an inability and/or decreased capacity of a cell, organ, and/or system to process and/or degrade trimethylamine and/or trimethylamine-N-oxide. In one embodiment, said inability or decreased capacity of a cell, organ, and/or system to process and/or degrade trimethylamine and/or trimethylamine-N-oxide is caused by the increased endogenous production of trimethylamine, e.g., increased endogenous production of trimethylamine by the intestinal microbiota in the gut. In another embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade trimethylamine is caused by the absence of, or a deficiency in, the expression or activity of the FM03 enzyme in the subject. In another embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade trimethylamine and/or trimethylamine-N-oxide is caused by an increased dietary intake of trimethylamine, trimethylamine N-oxide (TMAO), or trimethylamine- containing nutrients, such as choline, phosphatidylcholine, and/or carnitine). In one embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade trimethylamine results from an increase in the number of or activity of intestinal trimethylamine-producing microorganisms.

[0101] In one embodiment, a "disease in which trimethylamine is detrimental" or a "disorder in which trimethylamine is detrimental" or a "disorder involving the catabolism of trimethylamine" or "disease in which trimethylamine and/or trimethylamine-N-oxide is detrimental" or a "disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental" or a "disorder involving the catabolism of trimethylamine and/or

trimethylamine-N-oxide" is a disease or disorder involving the abnormal, e.g., increased, levels of trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) in a subject. In one embodiment, a disease or disorder in which trimethylamine is detrimental is

trimethylaminuria. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is a cardiovascular disease. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is kidney disease, such as chronic kidney disease. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is diabetes mellitus. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is insulin resistance. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is metabolic syndrome. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is nonalcoholic fatty liver disease. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is nonalcoholic steatohepatitis. In one embodiment, a subject having a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental exhibits a daily urinary excretion of a ratio of less than 85% of TMAO/(TMAO+TMA) (see, for example, Wevers et ah, 2008, Laboratory Guide to the Methods in Biochemical Genetics, Springer- Verlag; New York, 2008, p. 781-792; and Mackay et al, 2011, Clin. Biochem. Rev., 32(l):33-43), although other methods for diagnosing diseases or disorders associated in which trimethylamine and/or trimethylamine-N-oxide is detrimental are known in the art (see, for example, U.S. 2012/0157397, the entire contents of which are expressly

incorporated herein by reference).

[0102] As used herein, the terms "cardiovascular disease" or "cardiovascular disorder" are terms used to classify numerous conditions affecting the heart, heart valves, and vasculature (e.g., veins and arteries) of the body, and encompasses diseases and conditions including, but not limited to arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, congestive heart failure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonary embolism, primary hypertension, atrial fibrillation, stroke, transient ischemic attack, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque, acute coronary syndrome, acute ischemic attack, sudden cardiac death, peripheral vascular disease, coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease.

[0103] As used herein, the term "atherosclerosis" refers to a subset of cardiovascular disease that include atherosclerosis as a component or precursor to the particular type of cardiovascular disease and includes, without limitation, CAD, PAD, and cerebrovascular disease. Atherosclerosis is a chronic inflammatory response that occurs in the walls of arterial blood vessels, involves the formation of atheromatous plaques that can lead to narrowing ("stenosis") of the artery, and can eventually lead to partial or complete closure of the arterial opening and/or plaque ruptures. Thus, atherosclerosis include the consequences of atheromatous plaque formation and rupture including, without limitation, stenosis or narrowing of arteries, heart failure, aneurysm formation including aortic aneurysm, aortic dissection, and ischemic events such as myocardial infarction and stroke.

[0104] As used herein, the phrase "exogenous environmental condition" or

"exogenous environment signal" refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced. The phrase "exogenous environmental conditions" is meant to refer to the environmental conditions external to the engineered micororganism, but endogenous or native to the host subject environment. Thus, "exogenous" and "endogenous" may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some

embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut. In some embodiments, the exogenous environmental condition is a tissue- specific or disease- specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is specific to a trimethylamine catabolism enzyme disease, e.g. , trimethylaminuria . In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An "oxygen level-dependent promoter" or "oxygen level-dependent regulatory region" refers to a nucleic acid sequence to which one or more oxygen level- sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. In some embodiments, the exogenous environmental conditions stimulate the activity of an inducible promoter of the invention. In one embodiment, the inducible promoter of the invention is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the invention, for example, tetracycline.

[0105] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR (fumarate and nitrate reductase), ANR, and DNR. Corresponding FNR- responsive promoters, ANR (anaerobic nitrate respiration)-responsive promoters, and DNR (dissimilatory nitrate respiration regulator)-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1.

[0106] In a non-limiting example, a promoter (PfnrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic conditions by the global

transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS.

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

[0107] In some embodiments, the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS). In some embodiments, exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut. In some embodiments, the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules.

[0108] In some embodiments, the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example,

tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response). In some embodiments, the loss of exposure to an exogenous environmental condition (for example, in vivo) inhibits the activity of an inducible promoter, as the exogenous

environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut). "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.

[0109] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria do not contain lipopolysaccharides (LPS). In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to certain strains belonging to the genus Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus,

Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum,

Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis and Saccharomyces boulardii (Sonnenborn et al, 2009; Dinleyici et al, 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Nonpathogenic bacteria also include commensal bacteria, which are present in the indigenous microbiota of the gut. In one embodiment, the disclosure further includes non-pathogenic Saccharomyces, such as Saccharomyces boulardii. Naturally pathogenic bacteria may be genetically engineered to reduce or eliminate pathogenicity.

[0110] "Probiotic" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. In some embodiments, the probiotic bacteria are Gram-negative bacteria. In some embodiments, the probiotic bacteria are Gram- positive bacteria. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, certain strains belonging to the genus Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus,

Lactobacillus paracasei, and Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al, 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al, 2010; Olier et al, 2012; Nougayrede et al, 2006). Nonpathogenic 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. [0111] As used herein, the term "auxotroph" or "auxotrophic" refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient) to support its growth. An "auxotrophic modification" is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient.

[0112] As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA).

[0113] As used herein, the terms "modulate" and "treat" and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof. In another embodiment, "modulate" and "treat" refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "modulate" and "treat" refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g. , stabilization of a discernible symptom),

physiologically (e.g. , stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.

[0114] Those in need of treatment may include individuals already having a particular medical disease, as well as those at risk of having, or who may ultimately acquire the disease. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely receptiveness to treatment of a subject having the disease. Disorders in which trimethylamine and/or trimethylamine-N-oxide is detrimental, e.g., cardiovascular disease, kidney disease, or trimethylaminuria, may be caused by inborn genetic mutations for which there are no known cures. Diseases can also be secondary to other conditions, e.g., an intestinal disorder or a bacterial infection. Treating diseases in which trimethylamine and/or trimethylamine-N-oxide is detrimental may encompass reducing normal levels of

trimethylamine and/or trimethylamine-N-oxide, reducing excess levels of trimethylamine and/or trimethylamine-N-oxide, or eliminating trimethylamine and/or trimethylamine-N- oxide, and does not necessarily encompass the elimination of the underlying disease. [0115] As used herein, "payload" refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus. In some embodiments, the payload is a therapeutic payload, e.g. , a trimethylamine and/or

trimethylamine-N-oxide catabolic enzyme or a trimethylamine and/or trimethylamine-N- oxide transporter polypeptide. In some embodiments, the payload is a regulatory molecule, e.g. , a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments the payload comprises an antibiotic resistance gene or genes. In some embodiments, the payload is encoded by a gene, multiple genes, gene cassette, or an operon. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads.

[0116] As used herein, the term "polypeptide" includes "polypeptide" as well as "polypeptides," and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, "peptides," "dipeptides," "tripeptides, "oligopeptides," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three- dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term "peptide" or "polypeptide" may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.

[0117] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms "fragment," "variant," "derivative" and "analog" include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non- naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non- conservative amino acid substitutions, deletions or additions.

[0118] Polypeptides also include fusion proteins. As used herein, the term "variant" includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term "fusion protein" refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion

proteins. "Derivatives" include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the

corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or

substitutions: -Ala, Pro, Gly, Gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, He, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

[0119] As used herein, the term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention.

Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or

substitution(s). These may be naturally occurring variants as well as artificially designed ones.

[0120] As used herein the term "linker", "linker peptide" or "peptide linkers" or "linker" refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g. , that link two polypeptide domains. As used herein the term "synthetic" refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.

[0121] As used herein the term "codon-optimized" refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A "codon-optimized sequence" refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0122] As used herein, the terms "secretion system" or "secretion protein" refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g. , polypeptide from the microbial, e.g. , bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g. ,HlyBD. Non- limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the polypeptide to be secreted include a "secretion tag" of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some

embodiments, the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the lysosomal enzyme(s) into the extracellular milieu. In some embodiments, the secretion system involves the generation of a "leaky" or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g., selected from lpp, ompA, ompA, ompF, tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[0123] As used herein a "pharmaceutical composition" refers to a preparation of bacterial cells with other components such as a physiologically suitable carrier and/or excipient.

[0124] 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.

[0125] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[0126] 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 disease, e.g., a disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disease or condition associated with daily urinary excretion of a ratio of less than 85% of TMAO/(TMAO+TMA) (see, for example, Wevers et ah, 2008, Laboratory Guide to the Methods in Biochemical Genetics, Springer- Verlag; New York, 2008, p. 781-792; and Mackay et al, 2011, Clin. Biochem. Rev., 32(l):33-43), although other methods for diagnosing diseases or disorders associated in which trimethylamine and/or trimethylamine-N-oxide is detrimental are known in the art (see, for example, U.S. 2012/0157397, the entire contents of which are expressly incorporated herein by reference). 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.

[0127] As used herein, the term "bacteriostatic" or "cytostatic" refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of engineered bacterial cell of the disclosure.

[0128] As used herein, the term "bactericidal" refers to a molecule or protein which is capable of killing the engineered bacterial cell of the disclosure.

[0129] As used herein, the term "toxin" refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the engineered bacterial cell of the disclosure, or which is capable of killing the engineered bacterial cell of the disclosure. The term "toxin" is intended to include bacteriostatic proteins and bactericidal proteins. The term "toxin" is intended to include, but not limited to, lytic proteins, bacteriocins {e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term "anti-toxin" or "antitoxin," as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term antitoxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.

[0130] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0131] 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.

[0132] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Bacterial Strains

[0133] The disclosure provides a bacterial cell that comprises at least one

heterologous gene encoding at least one trimethylamine catabolism enzyme. In some embodiments, the bacterial cell is a non-pathogenic bacterial cell. In some embodiments, the bacterial cell is a commensal bacterial cell. In some embodiments, the bacterial cell is a probiotic bacterial cell.

[0134] In certain embodiments, the bacterial cell is selected from the group consisting of a Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis,

Clostridium butyricum, Clostridium scindens, Escherichia coli, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, and Oxalobacter formigenes bacterial cell. In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium animalis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one

embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell. In one

embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is a Clostridium scindens bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Oxalobacter formigenes bacterial cell. In another embodiment, the bacterial cell does not include Oxalobacter formigenes.

[0135] In one embodiment, the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.

[0136] In some embodiments, the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram- negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et ah, 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et ah, 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors {e.g., E. coli a-hemolysin, P- fimbrial adhesins) (Schultz, 2008), 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 ah, 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. It is commonly accepted that E. coli Nissle' s therapeutic efficacy and safety have convincingly been proven (Ukena et ah, 2007).

[0137] In one embodiment, the engineered bacterial cell does not colonize the subject having the disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental.

[0138] 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, e.g., a trimethylamine catabolism gene from Hyphomicrobium spp. can be expressed in Escherichia coli. Furthermore, genes from one or more different species can be introduced into one another, e.g., a gene from Lactobacillus plantarum or Methanobrevibacter smithii 3142 can be expressed in Escherichia coli.

[0139] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells.

[0140] In another aspect, the disclosure provides an engineered bacterial culture which comprises engineered bacterial cells.

[0141] In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette(s) are present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette(s) is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[0142] In some embodiments, the genetically engineered bacteria is an auxotroph or a conditional auxotroph. In one embodiment, the genetically engineered bacteria is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxo trophy, for example, they may be a AthyA and AdapA auxotroph.

[0143] In some embodiments, the genetically engineered bacteria further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of an promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD- In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

[0144] In some embodiments, the genetically engineered bacteria is an auxotroph and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.

[0145] In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette(s) are present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette(s) are present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[0146] The genetically engineered microorganisms, or programmed microorganisms, such as genetically engineered bacteria of the disclosure are capable of producing one or more enzymes for metabolizing trimethylamine and/or trimethylamine-N-oxide. Non- limiting examples of such enzymes and trimethylamine and/or trimethylamine-N-oxide metabolic pathways are described herein. In some aspects, the disclosure provides a bacterial cell that comprises one or more heterologous gene sequence(s) and/or gene cassette(s) encoding one or more trimethylamine catabolism enzyme(s) or other protein(s) that results in a decrease in levels of trimethylamine.

[0147] In certain embodiments, the genetically engineered bacteria are obligate anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are facultative anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are aerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram- positive bacteria. In some embodiments, the genetically engineered bacteria are Gram- positive bacteria and lack LPS. In some embodiments, the genetically engineered bacteria are Gram-negative bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and obligate anaerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and facultative anaerobic bacteria. In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some

embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria,

Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55,

Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT, Clostridium paraputrificum, Clostridium

pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, and Vibrio cholera. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei,

Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the the genetically engineered bacteria are selected from Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis,

Clostridium butyricum, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a

Bifidobacterium infantis bacterial cell. In one embodiment, the bacterial cell is a

Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell

[0148] In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors (e.g., E. coli - hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been used to treat ulcerative colitis in humans in vivo

(Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle' s therapeutic efficacy and safety have convincingly been proven (Ukena et al., 2007). [0149] In some embodiments, the genetically engineered bacteria are bacteria that naturally degrade TMA and/or TMAO. In some embodiments, th genetically engineered bacteria are Paracoccus or Hyphomicrobium spp, e.g. Hyphomicrobium X and Paracoccus T231.

[0150] 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.

Furthermore, genes from one or more different species can be introduced into one another, e.g., a trimethylamine catabolism gene from Hyphomicrobium spp. can be expressed in Escherichia coli. In some embodiments, the genes are codon optimized, e.g., for expression in E. coli. In one embodiment, the recombinant bacterial cell does not colonize the subject having the disorder. Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et ah, 2009). In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention.

[0151] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells disclosed herein.

[0152] In another aspect, the disclosure provides an engineered bacterial culture which comprises bacterial cells disclosed herein. In one aspect, the disclosure provides an engineered bacterial culture which reduces levels of trimethylamine (TMA) in the media of the culture. In one embodiment, the levels of trimethylamine (TMA) are reduced by about 50%, about 75%, or about 100% in the media of the cell culture. In another embodiment, the levels of trimethylamine (TMA) are reduced by about two-fold, three-fold, four-fold, fivefold, six- fold, seven- fold, eight-fold, nine-fold, or ten- fold in the media of the cell culture. In one embodiment, the levels of trimethylamine (TMA) are reduced below the limit of detection in the media of the cell culture.

[0153] In another aspect, the disclosure provides an engineered bacterial culture which comprises bacterial cells disclosed herein. In one aspect, the disclosure provides an engineered bacterial culture which reduces levels of TMAO in the media of the culture. In one embodiment, the levels of TMAO are reduced by about 50%, about 75%, or about 100% in the media of the cell culture. In another embodiment, the levels of TMAO are reduced by about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten- fold in the media of the cell culture. In one embodiment, the levels of TMAO are reduced below the limit of detection in the media of the cell culture.

[0154] In some embodiments of the above described genetically engineered bacteria, the gene encoding a trimethylamine catabolism enzyme is present on a plasmid in the bacterium and operatively linked on the plasmid to a promoter that is induced under low- oxygen or anaerobic conditions, such as any of the promoters disclosed herein. In other embodiments, the gene encoding a trimethylamine catabolism enzyme is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions, such as any of the promoters disclosed herein. In some embodiments of the above described genetically engineered bacteria, the gene encoding a trimethylamine catabolism enzyme is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under inflammatory conditions, such as any of the promoters disclosed herein. In other embodiments, the gene encoding a trimethylamine catabolism enzyme is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under inflammatory conditions, such as any of the promoters disclosed herein.

[0155] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding a trimethylamine catabolism enzyme is an auxotroph. In one

embodiment, the genetically engineered bacteria is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and Ml auxotroph. In some

embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[0156] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding a trimethylamine catabolism enzyme further comprise gene sequence(s) encoding a TMA and/or TMAO transporter which imports TMA and/or TMAO into the bacterial cell.

[0157] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding a trimethylamine catabolism enzyme further comprise gene sequence(s) encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein.

[0158] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding a trimethylamine catabolism enzyme further comprise gene sequence(s) encoding one or more antibiotic gene(s), such as any of the antibiotic genes disclosed herein. [0159] In some embodiments, the genetically engineered bacteria comprising a trimethylamine catabolism enzyme further comprise a kill- switch circuit, such as any of the kill- switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more

recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

[0160] In some embodiments, the genetically engineered bacteria is an auxotroph comprising gene sequence encoding a trimethylamine catabolism enzyme and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.

[0161] In some embodiments of the above described genetically engineered bacteria, the gene encoding a trimethylamine catabolism enzyme is present on a plasmid in the bacterium. In some embodiments, the gene encoding a trimethylamine catabolism enzyme is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding a trimethylamine transporter is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding a trimethylamine and/or trimethylamine-N-oxide transporter is present in the bacterial chromosome. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present on a plasmid in the bacterium. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome. Trimethylamine (TMA) Catabolism Enzymes

[0162] As used herein, the terms "trimethylamine catabolism enzyme", "TMA catabolism enzyme", "TMA and/or TMAO catabolism enzyme", and "trimethylamine and/or trimethylamine-N-oxide catabolism enzyme" refer to an enzyme involved in the processing, metabolism, degradation, or breakdown of trimethylamine to a non-toxic molecule, such as γ- glutamylmethylamide, or such as carbon dioxide and NADH (see, for example Fig. 1A and Fig. IB), or other non-toxic biproducts. In addition, the terms "trimethylamine catabolism enzyme", "TMA catabolism enzyme", "TMA and/or TMAO catabolism enzyme", and "trimethylamine and/or trimethylamine-N-oxide catabolism enzyme" also includes enzymes which catabolize TMAO, e.g., convert TMAO to TMA. Enzymes involved in the catabolism of trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) are well known to those of skill in the art. For example, in Hyphomicrobium and Paracoccus species, trimethylamine catabolism to γ-glutamylmethylamide or to carbon dioxide and NADH occurs using a several-step enzymatic process (see, for example, Meiberg and Harder, 1978, J. Gen.

Microbiol, 106:265-276; Kim et al, 2001, Arch. Microbiol, 176:271-277; and Kim et al, 2003, FEMS Microbiol. Letters, 225:263-269). For example, as demonstrated in Fig. 1A, trimethylamine is converted to dimethylamine by a trimethylamine dehydrogenase.

Dimethylamine is then converted to methylamine by a dimethylamine dehydrogenase. Next, methylamine and glutamate are converted to γ-glutamylmethylamide by a γ- glutamylmethylamide synthetase, and γ-glutamylmethylamide is converted to N- methylglutamate by an N-methylglutamate synthase. N-methylglutamate is then converted to glutamate and formaldehyde by an N-methyl-glutamate dehydrogenase. Formaldehyde is then either assimilated as a carbon source through the serine cycle or oxidized to carbon dioxide by a formaldehyde dehydrogenase and a formate dehydrogenase, and the ammonium produced is assimilated as a nitrogen source through the glutamine synthase/glutamate synthase pathway. However, other pathways to catabolize trimethylamine can also be utilized. For example, a methylamine-glutamate N-methyltransferase has been identified which converts methylamine and glutamate directly to N-methylglutamate (see, for example, Fig. IB and Shaw et al, 1966, J. Biol. Chem., 241:935-945).

[0163] The bacterial cells of the invention may comprise at least one heterologous gene encoding at least one trimethylamine catabolism enzyme and are capable of converting trimethylamine into γ-glutamylmethylamide, or into carbon dioxide and NADH (see, for example, Fig. 1A and Fig. IB) or other non-toxic byproduct(s). In some embodiments, the genetically engineered bacteria further comprise a trimethylamine (TMA) and/or TMAO transporter to facilitate the import of TMA and/or TMAO into the bacterial cell.

[0164] In one embodiment, the trimethylamine catabolism enzyme increases the rate of trimethylamine and/or trimethylamine-N-oxide catabolism in the cell or in the subject. In one embodiment, the trimethylamine and/or trimethylamine-N-oxide catabolism enzyme decreases the level of trimethylamine and/or trimethylamine-N-oxide in the cell or in the subject. In one embodiment, the trimethylamine catabolism enzyme decreases the level of trimethylamine N-oxide (TMAO) in the cell or in the subject.

[0165] In another embodiment, the trimethylamine catabolism enzyme increases the level of trimethylamine N-oxide (TMAO) in the cell or in the subject as compared to the level of trimethylamine (TMA) in the cell or in the subject. In another embodiment, the trimethylamine catabolism enzyme increases the level of carbon dioxide and NADH in the cell. In another embodiment, the trimethylamide catabolism enzyme increases the level of γ- glutamylmethylamide in the cell.

[0166] In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more enzymes involved in the catabolism of trimethylamine N-oxide (TMAO), e.g., an enzyme that catabilizes TMAO. In one embodiment, the enzyme is a TMAO reductase, which can convert TMAO to TMA. TMAO reductases from various bacterial species are well known in the art. In one embodiment, the TMAO reductase is from E coli. The TMAO reductase from E. coli can access TMAO in the periplasm. The torCA genes encode an inducible trimethylamine N-oxide (TMAO) reductase in E. coli K-12. The TMAO reductase complex consists of a periplasmic molybdenum containing subunit (TorA) and a membrane associated pentaheme c-type cytochrome (TorC). E. coli K-12 also contains a second TMAO reductase encoded by torZY. torZY has a low level of constitutive expression and is not induced by TMAO. In some embodiments, the genetically engineered bacteria comprise one or more TMAO reductases, e.g., TMAO reductases from E. coli. In some embodiments the TMAO reductases convert TMAO into TMA in the periplasm, and TMA is then further taken up into the the cytoplasm of the bacterial cell, where it is catabolized by one or more of the TMA catabolism enzymes described herein. In some embodiments the engineered bacteria do not require a TMAO transporter to take up TMAO. For example, if TMAO is reduced to TMA in the periplasm a TMAO transporter may not be required. In other embodiments, the engineered bacteria further comprise a TMAO

transporter to further improved the uptake of TMAO from the extracellular environment. [0167] Enzymes involved in the catabolism of trimethylamine and/or trimethylamine N-oxide may be expressed or modified in the bacteria in order to enhance catabolism of trimethylamine. Specifically, when at least one trimethylamine catabolism enzyme is expressed in the bacterial cells, the bacterial cells convert more trimethylamine into carbon dioxide and NADH when the catabolism enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when at least one trimethylamine catabolism enzyme is expressed in the bacterial cells, the bacterial cells convert more trimethylamine into γ-glutamylmethylamide when the catabolism enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the engineered bacteria comprising gene sequence encoding one or more

trimethylamine catabolism enzyme(s) can catabolize trimethylamine to treat disorders in which trimethylamine and/or trimethylamine-N-oxide is detrimental, including

cardiovascular diseases and disorders, chronic kidney disease, and trimethylaminuria. In some embodiments, the engineered bacteria comprising a heterologous gene encoding at least one trimethylamine catabolism enzyme can catabolize trimethylamine-N-oxide to treat disorders in which trimethylamine and/or trimethylamine-N-oxide is detrimental, including cardiovascular diseases and disorders, chronic kidney disease, and trimethylaminuria.

[0168] In one embodiment, the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s). In some embodiments, the invention provides a bacterial cell that comprising gene sequence encoding one or more trimethylamine catabolism enzyme(s) operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises gene sequence encoding one or more trimethylamine catabolism enzyme(s) from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding at least one trimethylamine catabolism enzyme. In yet another embodiment, the bacterial cell comprises at least one native gene encoding at least one trimethylamine catabolism enzyme, as well as at least one copy of at least one gene encoding at least one trimethylamine catabolism enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding at least one trimethylamine catabolism enzyme. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding at least one trimethylamine catabolism enzyme.

[0169] Multiple distinct trimethylamine catabolism enzymes are known in the art. In some embodiments, trimethylamine catabolism enzyme is encoded by at least one gene encoding at least one trimethylamine catabolism enzyme derived from a bacterial species. In some embodiments, at least one trimethylamine catabolism enzyme is encoded by a gene encoding at least one trimethylamine catabolism enzyme derived from a non-bacterial species. In some embodiments, at least one trimethylamine catabolism enzyme is encoded by a gene derived from a eukaryotic species, e.g., a yeast species or a plant species. In one embodiment, at least one trimethylamine catabolism enzyme is encoded by a gene derived from a human. In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to, Achromobacter parvulus, Acidomonas methanolica,

Aminobacter aminovorans, Ancylobacter aquaticus, Arthrobacter spp., Bacillus spp., such as Bacillus methanolicus, Beggiatoa alba, Ceriporiopsis subvermispora, Clostridium

carboxidivorans, Cupriavidus necator, Cupriavidus oxalaticus, Desulfovibrio desulfuricans, Escherichia coli, Glycine max, Glycine soja, Gottschalkia acidurici, Hyphomicrobium spp., Kloeckera spp., Komagataella pastrois, Lotus japonicas, Methylobacterium spp., such as Methylobacterium extorquens or Methylobacterium organophilum, Methylobacterium lusitanum, Methylobacterium oryzae, Methylobacterium salsuginis, Methylococcus spp., such as Methylococcus capsulatus, Methylomicrobium album, or Methylophaga spp., Methylocella silvestris, Methylophaga spp., such as Methylophaga marina or Methylophaga thalassica, Methylophilus methylotrophus, Methylosinus trichosporium, Methyloversatilis universalis, Methylovorus mays, Moraxella spp., Mycobacterium vaccae, Ogataea angusta, Ogataea pini, Paracoccus spp., such as Paracoccus dentrificans, Pisum sativum, Pseudomonas spp., such as Pseudomonas putida or Pseudomonas methylica, Rastrelliger kanagurta,

Rhodopseudomonas palustris, Thiobacillus spp., or Viqna radiate.

[0170] In another embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to Arabidopsis thaliana, Candida spp., such as Candida boidinii, Candida methanolica, or Candida methylica, Saccharaomyces cerevisiae, or Torulopsis Candida.

[0171] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to, Bifidobacterium, Bordetella, Bradyrhizobium, Burkholderia, Clostridium, Enterococcus, Escherichia, Eubacterium, Lactobacillus, Magneto spirillium, Mycobacterium, Neurospora, Oxalobacter, Ralstonia, Rhodopseudomonas, Shigella, Thermoplasma, and Thauera, e.g., Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bordatella bronchiseptica, Bordatella parapertussis, Burkholderia fungorum, Burkholderia xenovorans, Bradyrhizobium japonicum, Clostridium acetobutylicum, Clostridium difficile, Clostridium scindens, Clostridium sporogenes, Clostridium tentani, Enterococcus faecalis, Escherichia coli, Eubacterium lentum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Magneto spirillium magentotaticum, Mycobacterium avium, Mycobacterium

intracellular, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Neurospora crassa, Oxalobacter formigenes, Ralstonia eutropha, Ralstonia metallidurans, Rhodopseudomonas palustris, Shigella flexneri, Thermoplasma volcanium, and Thauera aromatica.

[0172] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme has been codon-optimized for use in the recombinant bacterial cell of the invention. In one embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme has been codon-optimized for use in Lactococcus. When the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed in the recombinant bacterial cells of the invention, the bacterial cells catabolize more

trimethylamine than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising at least one heterologous gene encoding at least one trimethylamine catabolism enzyme may be used to catabolize excess trimethylamine to treat a disorder in which trimethylamine is detrimental, such as cardiovascular disease, chronic kidney disease, or trimethylaminuria.

[0173] The present invention further comprises genes encoding functional fragments of at least one trimethylamine catabolism enzyme or functional variants of at least one trimethylamine catabolism enzyme. As used herein, the term "functional fragment thereof or "functional variant thereof of at least one trimethylamine catabolism enzyme relates to an element having qualitative biological activity in common with the wild-type trimethylamine catabolism enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated trimethylamine catabolism enzyme is one which retains essentially the same ability to catabolize trimethylamine as the

trimethylamine catabolism enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having trimethylamine catabolism enzyme activity may be truncated at the N-terminus or C-terminus and the retention of trimethylamine catabolism enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the recombinant bacterial cell of the invention comprises a heterologous gene encoding at least one trimethylamine catabolism enzyme functional variant. In another embodiment, the recombinant bacterial cell of the invention comprises a heterologous gene encoding at least one trimethylamine catabolism enzyme functional fragment.

[0174] Assays for testing the activity of at least one trimethylamine catabolism enzyme, at least one trimethylamine catabolism enzyme functional variant, or at least one trimethylamine catabolism enzyme functional fragment are well known to one of ordinary skill in the art. For example, trimethylamine catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous trimethylamine catabolism enzyme activity. Trimethylamine catabolism activity can be assessed by quantifying trimethylamine degradation in the culture media as described by Kim et ah, 2003, FEMS Microbiol. Letters, 225:263-269, the entire contents of which are expressly incorporated herein by reference. Alternatively, a mouse model of atherosclerosis can be used to assay trimethylamine catabolism activity in vivo as described by Gregory et ah, 2015, J. Biol. Chem., 290(9):5647-5660, the entire contents of which are expressly incorporated herein by reference.

[0175] As used herein, the term "percent (%) sequence identity" or "percent (%) identity," also including "homology," is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). [0176] The present invention encompasses genes encoding at least one trimethylamine catabolism enzyme comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, He, Asn, Gin, Phe, Cys, Pro, Trp, Tyr, Val).

[0177] In some embodiments, the gene encoding at least one trimethylamine catabolism enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the trimethylamine catabolism enzyme is isolated and inserted into the bacterial cell of the invention. In one embodiment, spontaneous mutants that arise that allow bacteria to grow on trimethylamine or trimethylamine N-oxide as the sole carbon source can be screened for and selected (see for example, Kim et ah, 2001, Arch. Microbiol., 176:271-277). The gene comprising the modifications described herein may be present on a plasmid or chromosome.

[0178] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is a trimethylamine dehydrogenase (EC 1.5.8.2). As used herein, the term "trimethylamine dehydrogenase" refers to an enzyme which coverts trimethylamine and water to dimethylamine and formaldehyde (see, for example, Fig. 1). In one embodiment, the trimethylamine dehydrogenase is from Hyphomicrobium spp. In another embodiment, the trimethylamine dehydrogenase is from Paracoccus spp. In another embodiment, the trimethylamine dehydrogenase is from Methylophilus methylotrophus. In another embodiment, the trimethylamine dehydrogenase is from Pseudomonas putida. In one embodiment, the trimethylamine dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: l. In another embodiment, the trimethylamine dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: l. In one embodiment, the trimethylamine dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: l. In one embodiment, the trimethylamine dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: l. In another embodiment, the trimethylamine dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: l. Accordingly, in one embodiment, the trimethylamine dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: l. In another embodiment, the trimethylamine dehydrogenase gene comprises the sequence of SEQ ID NO: l. In yet another embodiment the trimethylamine dehydrogenase gene consists of the sequence of SEQ ID NO: l.

[0179] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is a dimethylamine dehydrogenase (EC 1.5.8.1) (see also Yang et ah, 1995, Eur. J. Biochem., 232:264-271). As used herein, the term "dimethylamine dehydrogenase" refers to an enzyme which coverts dimethylamine and water to methylamine and formaldehyde (see, for example, Fig. 1). In one embodiment, the dimethylamine dehydrogenase is from Hyphomicrobium spp. In another embodiment, the dimethylamine dehydrogenase is from Paracoccus spp. In another embodiment, the dimethylamine dehydrogenase is from Pseudomonas putida. In one embodiment, the dimethylamine dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO:2. In another embodiment, the dimethylamine dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO:2. In one embodiment, the

dimethylamine dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO:2. In one embodiment, the dimethylamine dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO:2. In another embodiment, the dimethylamine dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:2. Accordingly, in one embodiment, the dimethylamine dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:2. In another embodiment, the dimethylamine dehydrogenase gene comprises the sequence of SEQ ID NO:2. In yet another embodiment the dimethylamine dehydrogenase gene consists of the sequence of SEQ ID NO:2. [0180] In one embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is a γ-glutamylmethylamine synthetase (EC 6.3.4.12). As used herein, the term "γ-glutamylmethylamine synthetase" refers to an enzyme which coverts ATP, L-glutamate, and methylamine to γ-glutamylmethylamide, ADP, and phosphate (see, for example, Fig. 1). In one embodiment, the γ-glutamylmethylamine synthetase is from Hyphomicrobium spp. In another embodiment, the γ-glutamylmethylamine synthetase is from Paracoccus spp. In another embodiment, the γ-glutamylmethylamine synthetase is from Methyloversatilis universalis. In another embodiment, the γ-glutamylmethylamine synthetase is from Methylovorus mays. In another embodiment, the γ-glutamylmethylamine synthetase is from Pseudomonas spp. In one embodiment, the γ-glutamylmethylamine synthetase gene has at least about 80% identity with the entire sequence of SEQ ID NO:3. In another embodiment, the γ-glutamylmethylamine synthetase gene has at least about 85% identity with the entire sequence of SEQ ID NO:3. In one embodiment, the γ- glutamylmethylamine synthetase gene has at least about 90% identity with the entire sequence of SEQ ID NO:3. In one embodiment, the γ-glutamylmethylamine synthetase gene has at least about 95% identity with the entire sequence of SEQ ID NO:3. In another embodiment, the γ-glutamylmethylamine synthetase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:3. Accordingly, in one embodiment, the γ-glutamylmethylamine synthetase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:3. In another embodiment, the γ- glutamylmethylamine synthetase gene comprises the sequence of SEQ ID NO:3. In yet another embodiment the γ-glutamylmethylamine synthetase gene consists of the sequence of SEQ ID NO:3.

[0181] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is an N-methylglutamate dehydrogenase (EC 1.5.99.5). As used herein, the term "N-methylglutamate dehydrogenase" refers to an enzyme which coverts N-methylglutamate and water into L-glutamate and formaldehyde (see, for example, Fig. 1). In one embodiment, the N-methylglutamate dehydrogenase is from Hyphomicrobium spp. In another embodiment, the N-methylglutamate dehydrogenase is from Paracoccus spp. In another embodiment, the N-methylglutamate dehydrogenase is from Aminobacter aminovorans. In another embodiment, the N-methylglutamate dehydrogenase is from Methylobacterium organophilum. In another embodiment, the N-methylglutamate dehydrogenase is from Methylocella silvestris. In another embodiment, the N- methylglutamate dehydrogenase is from Methylophaga spp., such as Methylophaga marina or Methylophaga thalassica. In another embodiment, the N-methylglutamate dehydrogenase is from Psuedomonas spp., such as Pseudomonas methylica. In one embodiment, the N- methylglutamate dehydrogenase comprises an A subunit, a B subunit, a C subunit, and a D subunit. In one embodiment, the A subunit of the N-methylglutamate dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 12. In another embodiment, the A subunit of the N-methylglutamate dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 12. In one embodiment, the A subunit of the N-methylglutamate dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 12. In one embodiment, the A subunit of the N-methylglutamate dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 12. In another embodiment, the A subunit of the N-methylglutamate dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 12. Accordingly, in one embodiment, the A subunit of the N-methylglutamate dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 12. In another embodiment, the A subunit of the N- methylglutamate dehydrogenase gene comprises the sequence of SEQ ID NO: 12. In yet another embodiment the A subunit of the N-methylglutamate dehydrogenase gene consists of the sequence of SEQ ID NO: 12. In one embodiment, the B subunit of the N-methylglutamate dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 13. In another embodiment, the B subunit of the N-methylglutamate dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 13. In one embodiment, the B subunit of the N-methylglutamate dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 13. In one embodiment, the B subunit of the N-methylglutamate dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 13. In another embodiment, the B subunit of the N- methylglutamate dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 13. Accordingly, in one embodiment, the B subunit of the N-methylglutamate dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 13. In another embodiment, the B subunit of the N-methylglutamate dehydrogenase gene comprises the sequence of SEQ ID NO: 13. In yet another embodiment the B subunit of the N-methylglutamate dehydrogenase gene consists of the sequence of SEQ ID NO: 13. In one embodiment, the C subunit of the N- methylglutamate dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 14. In another embodiment, the C subunit of the N-methylglutamate dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 14. In one embodiment, the C subunit of the N-methylglutamate dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 14. In one embodiment, the C subunit of the N-methylglutamate dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 14. In another embodiment, the C subunit of the N-methylglutamate dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 14. Accordingly, in one embodiment, the C subunit of the N-methylglutamate dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 14. In another embodiment, the C subunit of the N-methylglutamate dehydrogenase gene comprises the sequence of SEQ ID NO: 14. In yet another embodiment the C subunit of the N-methylglutamate

dehydrogenase gene consists of the sequence of SEQ ID NO: 14. In one embodiment, the D subunit of the N-methylglutamate dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 15. In another embodiment, the D subunit of the N- methylglutamate dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 15. In one embodiment, the D subunit of the N-methylglutamate

dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 15. In one embodiment, the D subunit of the N-methylglutamate dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 15. In another embodiment, the D subunit of the N-methylglutamate dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 15. Accordingly, in one embodiment, the D subunit of the N-methylglutamate dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 15. In another embodiment, the D subunit of the N-methylglutamate dehydrogenase gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the D subunit of the N- methylglutamate dehydrogenase gene consists of the sequence of SEQ ID NO: 15.

[0182] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is a methanol dehydrogenase (EC 1.1.1.244). As used herein, the term "methanol dehydrogenase" refers to an enzyme which coverts methanol to formaldehyde. In one embodiment, the methanol dehydrogenase is from Hyphomicrobium spp. In another embodiment, the methanol dehydrogenase is from Paracoccus spp. In another embodiment, the methanol dehydrogenase is from Acidomonas methanolica, Bacillus methanolicus, Beggiatoa alba, Methylobacterium lusitanum, Methylobacterium oryzae, Methylobacterium salsuginis, Methylococcus capsulatus, Methylomicrobium album, or Methylophaga spp. In one embodiment, the methanol dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 17. In another embodiment, the methanol dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 17. In one embodiment, the methanol dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 17. In one embodiment, the methanol dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 17. In another embodiment, the methanol dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 17. Accordingly, in one embodiment, the methanol dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 17. In another embodiment, the methanol dehydrogenase gene comprises the sequence of SEQ ID NO: 17. In yet another embodiment the methanol dehydrogenase gene consists of the sequence of SEQ ID NO: 17.

[0183] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is a formaldehyde dehydrogenase (EC 1.2.1.46). As used herein, the term "formaldehyde dehydrogenase" refers to an enzyme which coverts formaldehyde and water into formate (see, for example, Fig. 1). In one embodiment, the formaldehyde dehydrogenase is from Hyphomicrobium spp. In another embodiment, the formaldehyde dehydrogenase is from Paracoccus spp. In another embodiment, the formaldehyde dehydrogenase is from Aminobacter aminovorans. In another embodiment, the formaldehyde dehydrogenase is from Arthrobacter spp. In another embodiment, the formaldehyde dehydrogenase is from Komagataella pastrois. In another embodiment, the formaldehyde dehydrogenase is from Methylococcus spp., such as Methylococcus capsulatus. In another embodiment, the formaldehyde dehydrogenase is from Methylophilus

methylotrophus. In another embodiment, the formaldehyde dehydrogenase is from

Pseudomonas putida. In another embodiment, the formaldehyde dehydrogenase is from Ogataea angusta. In another embodiment, the formaldehyde dehydrogenase is from

Rastrelliger kanagurta. In one embodiment, the formaldehyde dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 16. In another embodiment, the formaldehyde dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 16. In one embodiment, the formaldehyde dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 16. In one embodiment, the formaldehyde dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 16. In another embodiment, the formaldehyde dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 16. Accordingly, in one embodiment, the formaldehyde dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 16. In another embodiment, the formaldehyde dehydrogenase gene comprises the sequence of SEQ ID NO: 16. In yet another embodiment the formaldehyde dehydrogenase gene consists of the sequence of SEQ ID NO: 16.

[0184] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is a formate dehydrogenase (EC 1.2.1.2). As used herein, the term "formate dehydrogenase" refers to an enzyme which coverts formate and NAD⁺ into carbon dioxide and NADH (see, for example, Fig. 1). In one embodiment, the formate dehydrogenase is from Hyphomicrobium spp. In another embodiment, the formate dehydrogenase is from Paracoccus spp. In another embodiment, the formate dehydrogenase is from Achromobacter parvulus. In another embodiment, the formate dehydrogenase is from Ancylobacter aquaticus. In another embodiment, the formate dehydrogenase is from

Arabidopsis thaliana. In another embodiment, the formate dehydrogenase is from

Arthrobacter spp. In another embodiment, the formate dehydrogenase is from Bacillus spp. In another embodiment, the formate dehydrogenase is from Candida spp., such as Candida boidinii, Candida methanolica, or Candida methylica. In another embodiment, the formate dehydrogenase is from Ceriporiopsis subvermispora. In another embodiment, the formate dehydrogenase is from Clostridium carboxidivorans. In another embodiment, the formate dehydrogenase is from Cupriavidus necator or Cupriavidus oxalaticus. In another embodiment, the formate dehydrogenase is from Desulfovibrio desulfuricans. In another embodiment, the formate dehydrogenase is from Escherichia coli. In another embodiment, the formate dehydrogenase is from Enterobacter cloacae. In another embodiment, the formate dehydrogenase is from Glycine max or Glycine soja. In another embodiment, the formate dehydrogenase is from Gottschalkia acidurici. In another embodiment, the formate dehydrogenase is from Kloeckera spp. In another embodiment, the formate dehydrogenase is from Komagataella pastoris. In another embodiment, the formate dehydrogenase is from Lotus japonicus. In another embodiment, the formate dehydrogenase is from

Methylobacterium spp., such as Methylobacterium extorquens or Methylobacterium organophilum. In another embodiment, the formate dehydrogenase is from Methylococcus capsulatas. In another embodiment, the formate dehydrogenase is from Methylophilus methylotrophus. In another embodiment, the formate dehydrogenase is from Methylosinus trichosporium. In another embodiment, the formate dehydrogenase is from Moraxella spp. In another embodiment, the formate dehydrogenase is from Mycobacterium vaccae. In another embodiment, the formate dehydrogenase is from Ogataea angusta. In another embodiment, the formate dehydrogenase is from Ogataea pini. In another embodiment, the formate dehydrogenase is from Paracoccus spp., such as Paracoccus dentrificans. In another embodiment, the formate dehydrogenase is from Pisum sativum. In another embodiment, the formate dehydrogenase is from Pseudomonas spp., such as Pseudomonas methylica. In another embodiment, the formate dehydrogenase is from Rhodopseudomonas palustris. In another embodiment, the formate dehydrogenase is from Saccharaomyces cerevisiae. In another embodiment, the formate dehydrogenase is from Thiobacillus spp. In another embodiment, the formate dehydrogenase is from Torulopsis Candida. In another

embodiment, the formate dehydrogenase is from Viqna radiate. In one embodiment, the formate dehydrogenase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 18. In another embodiment, the formate dehydrogenase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 18. In one embodiment, the formate dehydrogenase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 18. In one embodiment, the formate dehydrogenase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 18. In another embodiment, the formate

dehydrogenase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 18. Accordingly, in one embodiment, the formate dehydrogenase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 18. In another embodiment, the formate dehydrogenase gene comprises the sequence of SEQ ID NO: 18. In yet another embodiment the formate dehydrogenase gene consists of the sequence of SEQ ID NO: 18.

[0185] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is a methylamine-glutamate N-methyltransferase (EC 2.1.1.21). As used herein, the term "methylamine-glutamate N-methyltransferase" refers to an enzyme which coverts methylamine and L-glutamate to N-methyl-L-glutamate and NH₃ (see, for example, Fig. IB). In one embodiment, the methylamine-glutamate N- methyltransferase is from Hyphomicrobium spp. In another embodiment, the methylamine- glutamate N-methyltransferase is from Paracoccus spp. In another embodiment, the methylamine-glutamate N-methyltransferase is from Methyloversatilis universalis. In another embodiment, the methylamine-glutamate N-methyltransferase is from Psuedomonas spp. In one embodiment, the methylamine-glutamate N-methyltransferase comprises an A subunit, a B subunit, and a C subunit. In one embodiment, the A subunit of the methylamine- glutamate N-methyltransferase gene has at least about 80% identity with the entire sequence of SEQ ID NO:9. In another embodiment, the A subunit of the methylamine-glutamate N- methyltransferase gene has at least about 85% identity with the entire sequence of SEQ ID NO:9. In one embodiment, the A subunit of the methylamine-glutamate N-methyltransferase gene has at least about 90% identity with the entire sequence of SEQ ID NO:9. In one embodiment, the A subunit of the methylamine-glutamate N-methyltransferase gene has at least about 95% identity with the entire sequence of SEQ ID NO:9. In another embodiment, the A subunit of the methylamine-glutamate N-methyltransferase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:9. Accordingly, in one embodiment, the A subunit of the methylamine-glutamate N-methyltransferase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:9. In another embodiment, the A subunit of the methylamine-glutamate N-methyltransferase gene comprises the sequence of SEQ ID NO:9. In yet another embodiment the A subunit of the methylamine-glutamate N-methyltransferase gene consists of the sequence of SEQ ID NO:9. In one embodiment, the B subunit of the methylamine-glutamate N-methyltransferase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 10. In another embodiment, the B subunit of the methylamine-glutamate N-methyltransferase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 10. In one embodiment, the B subunit of the methylamine-glutamate N-methyltransferase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 10. In one embodiment, the B subunit of the methylamine-glutamate N-methyltransferase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 10. In another embodiment, the B subunit of the

methylamine-glutamate N-methyltransferase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 10. Accordingly, in one embodiment, the B subunit of the methylamine-glutamate N-methyltransferase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 10. In another embodiment, the B subunit of the methylamine-glutamate N-methyltransferase gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the B subunit of the methylamine- glutamate N-methyltransferase gene consists of the sequence of SEQ ID NO: 10. In one embodiment, the C subunit of the methylamine-glutamate N-methyltransferase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 11. In another embodiment, the C subunit of the methylamine-glutamate N-methyltransferase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 11. In one embodiment, the C subunit of the methylamine-glutamate N-methyltransferase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 11. In one embodiment, the C subunit of the methylamine- glutamate N-methyltransferase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 11. In another embodiment, the C subunit of the methylamine-glutamate N- methyltransferase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 11. Accordingly, in one embodiment, the C subunit of the methylamine-glutamate N-methyltransferase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 11. In another embodiment, the C subunit of the methylamine-glutamate N-methyltransferase gene comprises the sequence of SEQ ID NO: 11. In yet another embodiment the C subunit of the methylamine-glutamate N- methyltransferase gene consists of the sequence of SEQ ID NO: 11.

[0186] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is an N-methylglutamate synthase (EC 2.1.1.21). As used herein, the term "N-methylglutamate synthase" refers to an enzyme which coverts γ- glutamylmethylamide to N-methylglutamate and NH₃ (see, for example, Fig. 1A). In one embodiment, the N-methylglutamate synthase is from Hyphomicrobium spp. In another embodiment, the N-methylglutamate synthase is from Paracoccus spp. In another embodiment, the N-methylglutamate synthase is from Methyloversatilis universalis. In another embodiment, the N-methylglutamate synthase is from Pseudomonas spp. In one embodiment, the N-methylglutamate synthase comprises an A subunit, a B subunit, and a C subunit. In one embodiment, the A subunit of the N-methylglutamate synthase gene has at least about 80% identity with the entire sequence of SEQ ID NO:9. In another embodiment, the A subunit of the N-methylglutamate synthase gene has at least about 85% identity with the entire sequence of SEQ ID NO:9. In one embodiment, the A subunit of the N- methylglutamate synthase gene has at least about 90% identity with the entire sequence of SEQ ID NO:9. In one embodiment, the A subunit of the N-methylglutamate synthase gene has at least about 95% identity with the entire sequence of SEQ ID NO:9. In another embodiment, the A subunit of the N-methylglutamate synthase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:9. Accordingly, in one embodiment, the A subunit of the N-methylglutamate synthase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:9. In another

embodiment, the A subunit of the N-methylglutamate synthase gene comprises the sequence of SEQ ID NO:9. In yet another embodiment the A subunit of the N-methylglutamate synthase gene consists of the sequence of SEQ ID NO:9. In one embodiment, the B subunit of the N-methylglutamate synthase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 10. In another embodiment, the B subunit of the N- methylglutamate synthase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 10. In one embodiment, the B subunit of the N-methylglutamate synthase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 10. In one embodiment, the B subunit of the N-methylglutamate synthase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 10. In another embodiment, the B subunit of the N-methylglutamate synthase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 10. Accordingly, in one embodiment, the B subunit of the N-methylglutamate synthase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 10. In another embodiment, the B subunit of the N- methylglutamate synthase gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the B subunit of the N-methylglutamate synthase gene consists of the sequence of SEQ ID NO: 10. In one embodiment, the C subunit of the N-methylglutamate synthase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 11. In another embodiment, the C subunit of the N-methylglutamate synthase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 11. In one embodiment, the C subunit of the N-methylglutamate synthase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 11. In one embodiment, the C subunit of the N-methylglutamate synthase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 11. In another embodiment, the C subunit of the N-methylglutamate synthase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 11. Accordingly, in one embodiment, the C subunit of the N-methylglutamate synthase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 11. In another embodiment, the C subunit of the N-methylglutamate synthase gene comprises the sequence of SEQ ID NO: 11. In yet another embodiment the C subunit of the N-methylglutamate synthase gene consists of the sequence of SEQ ID NO: 11.

[0187] In one embodiment, the bacteria comprise a TMA catabolism cassette(s) encoding one or more Trimethylamine dehydrogenase polypeptide(s), including, but not limited to, Trimethylamine dehydrogenase from Methylophilus methylotrophus . In one embodiment the one or more Trimethylamine dehydrogenase polypeptide(s) have at least about 80% identity with SEQ ID NO: 47. In another embodiment, the one or more

Trimethylamine dehydrogenase polypeptide(s) have at least about 85% identity with SEQ ID NO: 47. In one embodiment, the one or more Trimethylamine dehydrogenase polypeptide(s) have at least about 90% identity with SEQ ID NO: 47. In one embodiment, the one or more Trimethylamine dehydrogenase polypeptide(s) have at least about 95% identity with SEQ ID NO: 47. In another embodiment, the one or more Trimethylamine dehydrogenase polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47. Accordingly, in one embodiment, the one or more Trimethylamine dehydrogenase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47. In another embodiment, the one or more Trimethylamine dehydrogenase polypeptide(s) comprise the sequence of SEQ ID NO: 47. In yet another embodiment, the one or more Trimethylamine dehydrogenase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by engineered bacteria consist of the sequence of SEQ ID NO: 47.

[0188] In one embodiment, the bacteria comprise a TMA catabolism cassette(s) encoding one or more Dimethylamine dehydrogenase polypeptide(s), including but not limited to, Dimethylamine dehydrogenase from Hyphomicrobium dentrificans. In one embodiment the one or more Dimethylamine dehydrogenase polypeptide(s) have at least about 80% identity with SEQ ID NO: 48. In another embodiment, the one or more

Dimethylamine dehydrogenase polypeptide(s) have at least about 85% identity with SEQ ID NO: 48. In one embodiment, the one or more Dimethylamine dehydrogenase polypeptide(s) have at least about 90% identity with SEQ ID NO: 48. In one embodiment, the one or more Dimethylamine dehydrogenase polypeptide(s) have at least about 95% identity with SEQ ID NO: 48. In another embodiment, the one or more Dimethylamine dehydrogenase polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. Accordingly, in one embodiment, the one or more Dimethylamine dehydrogenase

polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the enginerred bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. In another embodiment, the one or more Dimethylamine dehydrogenase

polypeptide(s) comprise the sequence of SEQ ID NO: 48. In yet another embodiment the one or more Dimethylamine dehydrogenase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria consist of the sequence of SEQ ID NO: 48.

[0189] In one embodiment, the bacteria comprise a TMA catabolism gene(s) or gene cassette(s) encoding one or more γ-glutamylmethylamine synthetase polypeptides, including but not limited to, γ-glutamylmethylamine synthetase from Methyloversatilis universalis. In one embodiment, the one or more γ-glutamylmethylamine synthetase polypeptide(s) have at least about 80% identity with SEQ ID NO: 49. In another embodiment, the one or more γ- glutamylmethylamine synthetase polypeptide(s) have at least about 85% identity with SEQ ID NO: 49. In one embodiment, the one or more γ-glutamylmethylamine synthetase polypeptide(s) have at least about 90% identity with SEQ ID NO: 49. In one embodiment, the one or more γ-glutamylmethylamine synthetase polypeptide(s) have at least about 95% identity with SEQ ID NO: 49. In another embodiment, the one or more γ- glutamylmethylamine synthetase polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. Accordingly, in one embodiment, the one or more γ- glutamylmethylamine synthetase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. In another embodiment, the one or more γ- glutamylmethylamine synthetase polypeptide(s) comprise the sequence of SEQ ID NO: 49. In yet another embodiment, the one or more γ-glutamylmethylamine synthetase

polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 49.

[0190] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more N-methylglutamate synthase subunit A polypeptide(s), including but not limited to, N-methylglutamate synthase subunit Afrom Meihyioversaiilis universalis. In one embodiment the one or more N-methylglutamate synthase subunit A polypeptide(s) have at least about 80% identity with SEQ ID NO: 50. In another

embodiment, the one or more N-methylglutamate synthase subunit A polypeptide(s) have at least about 85% identity with SEQ ID NO: 50. In one embodiment, the one or more N- methylglutamate synthase subunit A polypeptide(s) have at least about 90% identity with SEQ ID NO: 50. In one embodiment, the one or more N-methylglutamate synthase subunit A polypeptide(s) have at least about 95% identity with SEQ ID NO: 50. In another embodiment, the one or more N-methylglutamate synthase subunit A polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 50. Accordingly, in one embodiment, the one or more N-methylglutamate synthase subunit A polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 50. In another embodiment, the one or more N-methylglutamate synthase subunit A polypeptide(s) comprise the sequence of SEQ ID NO: 50. In yet another embodiment, the one or more N- methylglutamate synthase subunit A polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 50.

[0191] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more N-methylglutamate synthase subunit B polypeptide(s), including but not limited to, N-methylglutamate synthase subunit B from Methyloversatilis universalis. In one embodiment, the one or more N-methylglutamate synthase subunit B polypeptide(s) have at least about 80% identity with SEQ ID NO: 51. In another

embodiment, the one or more N-methylglutamate synthase subunit B polypeptide(s) have at least about 85% identity with SEQ ID NO: 51. In one embodiment, the one or more N- methylglutamate synthase subunit B polypeptide(s) have at least about 90% identity with SEQ ID NO: 51. In one embodiment, the one or more N-methylglutamate synthase subunit B polypeptide(s) have at least about 95% identity with SEQ ID NO: 51. In another

embodiment, the one or more N-methylglutamate synthase subunit B polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 51. Accordingly, in one embodiment, the one or more N-methylglutamate synthase subunit B polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 51. In another embodiment, the one or more N-methylglutamate synthase subunit B polypeptide(s) comprise the sequence of SEQ ID NO: 51. In yet another embodiment, the one or more N- methylglutamate synthase subunit B polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 51.

[0192] In one embodiment, the bacteria comprises TMA catabolism gene(s) or gene cassette(s) encoding one or more N-methylglutamate synthase subunit C polypeptide(s), including but not limited to, N-methylglutamate synthase subunit C from Methyloversatilis universalis. In one embodiment, the one or more N-methylglutamate synthase subunit C polypeptide(s) have at least about 80% identity with SEQ ID NO: 52. In another

embodiment, the one or more N-methylglutamate synthase subunit C polypeptide(s) have at least about 85% identity with SEQ ID NO: 52. In one embodiment, the one or more N- methylglutamate synthase subunit C polypeptide(s) have at least about 90% identity with SEQ ID NO: 52. In one embodiment, the one or more N-methylglutamate synthase subunit C polypeptide(s) have at least about 95% identity with SEQ ID NO: 52. In another

embodiment, the one or more N-methylglutamate synthase subunit C polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 52. Accordingly, in one embodiment, the one or more N-methylglutamate synthase subunit C polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 52. In another embodiment, the one or more N-methylglutamate synthase subunit C polypeptide(s) comprise the sequence of SEQ ID NO: 52. In yet another embodiment, the one or more N- methylglutamate synthase subunit C polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 52.

[0193] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more N-methylglutamate dehydrogenase subunit A

polypeptide(s), including but not limited to N-methylglutamate dehydrogenase subunit A from Methyloversatilis universalis. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit A polypeptide(s) have at least about 80% identity with SEQ ID NO: 53. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit A polypeptide(s) have at least about 85% identity with SEQ ID NO: 53. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit A polypeptide(s) have at least about 90% identity with SEQ ID NO: 53. In one embodiment, the one or more N- methylglutamate dehydrogenase subunit A polypeptide(s) have at least about 95% identity with SEQ ID NO: 53. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit A polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. Accordingly, in one embodiment, the one or more N-methylglutamate dehydrogenase subunit A polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. In another embodiment, the one or more N- methylglutamate dehydrogenase subunit A polypeptide(s) comprise the sequence of SEQ ID NO: 53. In yet another embodiment , the one or more N-methylglutamate dehydrogenase subunit A polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 53.

[0194] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more N-methylglutamate dehydrogenase subunit B

polypeptide(s), including but not limited to, N-methylglutamate dehydrogenase subunit B from Methyloversatilis universalis. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit B polypeptide(s) have at least about 80% identity with SEQ ID NO: 54. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit B polypeptide(s) have at least about 85% identity with SEQ ID NO: 54. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit B polypeptide(s) have at least about 90% identity with SEQ ID NO: 54. In one embodiment, the one or more N- methylglutamate dehydrogenase subunit B polypeptide(s) have at least about 95% identity with SEQ ID NO: 54. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit B polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. Accordingly, in one embodiment, the one or more N-methylglutamate dehydrogenase subunit B polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. In another embodiment, the one or more N- methylglutamate dehydrogenase subunit B polypeptide(s) comprise the sequence of SEQ ID NO: 54. In yet another embodiment, the one or more N-methylglutamate dehydrogenase subunit B polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 54. [0195] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) that encode one or more N-methylglutamate dehydrogenase subunit C

polypeptides, including but not limited to, N-methylglutamate dehydrogenase subunit C from Methyloversatilis universalis. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit C polypeptide(s) have at least about 80% identity with SEQ ID NO:

55. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit C polypeptide(s) have at least about 85% identity with SEQ ID NO: 55. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit C polypeptide(s) have at least about 90% identity with SEQ ID NO: 55. In one embodiment, the one or more N- methylglutamate dehydrogenase subunit C polypeptide(s) have at least about 95% identity with SEQ ID NO: 55. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit C polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. Accordingly, in one embodiment, the one or more N-methylglutamate dehydrogenase subunit C polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. In another embodiment, the one or more N- methylglutamate dehydrogenase subunit C polypeptide(s) comprise the sequence of SEQ ID NO: 55. In yet another embodiment, the one or more N-methylglutamate dehydrogenase subunit C polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 55.

[0196] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more N-methylglutamate dehydrogenase subunit D

polypeptide(s), including but not limited to, N-methylglutamate dehydrogenase subunit D from Methyloversatilis universalis. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit D polypeptide(s) have at least about 80% identity with SEQ ID NO:

56. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit D polypeptide(s) have at least about 85% identity with SEQ ID NO: 56. In one embodiment, the one or more N-methylglutamate dehydrogenase subunit D polypeptide(s) have at least about 90% identity with SEQ ID NO: 56. In one embodiment, the one or more N- methylglutamate dehydrogenase subunit D polypeptide(s) have at least about 95% identity with SEQ ID NO: 56. In another embodiment, the one or more N-methylglutamate dehydrogenase subunit D polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. Accordingly, in one embodiment, the one or more N-methylglutamate dehydrogenase subunit D polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. In another embodiment, the one or more N- methylglutamate dehydrogenase subunit D polypeptide(s) omprise the sequence of SEQ ID NO: 56. In yet another embodiment, the one or more N-methylglutamate dehydrogenase subunit D polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 56.

[0197] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more formaldehyde dehydrogenase polypeptide(s), including but not limited to, formaldehyde dehydrogenase from Burkholderia sp.. In one embodiment the one or more formaldehyde dehydrogenase polypeptide(s) have at least about 80% identity with SEQ ID NO: 57. In another embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) have at least about 85% identity with SEQ ID NO: 57. In one embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) have at least about 90% identity with SEQ ID NO: 57. In one embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) have at least about 95% identity with SEQ ID NO: 57. In another

embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. Accordingly, in one embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. In another embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) comprise the sequence of SEQ ID NO: 57. In yet another embodiment, the one or more formaldehyde dehydrogenase polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 57.

[0198] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more methanol dehydrogenase polypeptide(s), including but not limited to, methanol dehydrogenase from Bacillus methanolicus . In one embodiment, the one or more methanol dehydrogenase polypeptide(s) have at least about 80% identity with SEQ ID NO: 58. In another embodiment, the one or more methanol dehydrogenase polypeptide(s) have at least about 85% identity with SEQ ID NO: 58. In one embodiment, the one or more methanol dehydrogenase polypeptide(s) have at least about 90% identity with SEQ ID NO: 58. In one embodiment, the one or more methanol dehydrogenase polypeptide(s) have at least about 95% identity with SEQ ID NO: 58. In another embodiment, the one or more methanol dehydrogenase polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. Accordingly, in one embodiment, the one or more methanol dehydrogenase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. In another embodiment, the one or more methanol

dehydrogenase polypeptide(s) comprise the sequence of SEQ ID NO: 58. In yet another embodiment, the one or more methanol dehydrogenase polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 58.

[0199] In one embodiment, the bacteria comprise TMA catabolism gene(s) or gene cassette(s) encoding one or more formate dehydrogenase polypeptide(s), including but not limited to, formate dehydrogenase from Enterobacter cloacae. In one embodiment the one or more formate dehydrogenase polypeptide(s) have at least about 80% identity with SEQ ID NO: 59. In another embodiment, the one or more formate dehydrogenase polypeptide(s) have at least about 85% identity with SEQ ID NO: 59. In one embodiment, the one or more formate dehydrogenase polypeptide(s) have at least about 90% identity with SEQ ID NO: 59. In one embodiment, the one or more formate dehydrogenase polypeptide(s) have at least about 95% identity with SEQ ID NO: 59. In another embodiment, the one or more formate dehydrogenase polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. Accordingly, in one embodiment, the one or more formate dehydrogenase polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. In another embodiment, the one or more formate dehydrogenase polypeptide(s) comprise the sequence of SEQ ID NO: 59. In yet another embodiment the one or more formate dehydrogenase polypeptide(s) encoded by the TMA catabolism cassette(s) and expressed by the genetically engineered bacteria consist of the sequence of SEQ ID NO: 59.

[0200] In one embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, and γ- glutamylmethylamide synthetase. In one embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ- glutamylmethylamide synthetase, N-methylglutamate synthetase, and N-methylglutamate dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ- glutamylmethylamide synthetase, N-methylglutamate synthetase, and N-methylglutamate dehydrogenase, and methanol dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, N-methylglutamate synthetase, N- methylglutamate dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, N-methylglutamate synthetase, N-methylglutamate dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol dehydrogenase.

[0201] In one embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine-glutamate N-methyltransferase, and N-methylglutamate dehydrogenase. In one embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism

enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine

dehydrogenase, dimethylamine dehydrogenase, methylamine-glutamate N-methyltransferase, N-methylglutamate dehydrogenase, and methanol dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine

dehydrogenase, dimethylamine dehydrogenase, methylamine-glutamate N-methyltransferase, N-methylglutamate dehydrogenase, formaldehyde dehydrogenase, and formate

dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine- glutamate N-methyltransferase, N-methylglutamate dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol dehydrogenase. [0202] In one embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase and dimethylamine dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, and methylamine-glutamate N- methyltransferase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine- glutamate N-methyltransferase, and N-methylglutamate dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine-glutamate N-methyltransferase, N-methylglutamate dehydrogenase, and formaldehyde dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine-glutamate N-methyltransferase, N-methylglutamate dehydrogenase, formaldehyde dehydrogenase, and formate

dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, methylamine- glutamate N-methyltransferase, N-methylglutamate dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol dehydrogenase.

[0203] In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, and γ- glutamylmethylamide synthetase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, and a N-methylglutamate synthase. In another embodiment, the bacteria comprise gene sequence encoding one or more

trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, a N-methylglutamate synthase, and an N-methylglutamate dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more

trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, a N-methylglutamate synthase, an N-methylglutamate dehydrogenase, and a formaldehyde dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ- glutamylmethylamide synthetase, a N-methylglutamate synthase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, and a formate dehydrogenase. In another embodiment, the bacteria comprise gene sequence encoding one or more trimethylamine catabolism enzyme(s), wherein the trimethylamine catabolism enzymes are trimethylamine dehydrogenase, dimethylamine dehydrogenase, γ-glutamylmethylamide synthetase, a N- methylglutamate synthase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, a formate dehydrogenase, and a methanol dehydrogenase.

[0204] In one embodiment, the at least one gene encoding the at least one

trimethylamine catabolism enzyme is operably linked to a directly inducible promoter. In another embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is operably linked to an indirectly inducible promoter. In one embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is operably linked to a promoter that differs from its natural promoter.

[0205] In some embodiments, the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed under the control of a constitutive promoter. In another embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed under the control of an inducible promoter. In some embodiments, the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the at least one gene encoding the at least one trimethylamine catabolism enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.

[0206] The at least one gene encoding the at least one trimethylamine catabolism enzyme may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is located on a plasmid in the bacterial cell. In another embodiment, the at least one gene encoding the at least one trimethylamine catabolism enzyme is located in the

chromosome of the bacterial cell. In yet another embodiment, a native copy of the at least one gene encoding the at least one trimethylamine catabolism enzyme is located in the chromosome of the bacterial cell, and at least one gene encoding at least one trimethylamine catabolism enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the at least one gene encoding the at least one trimethylamine catabolism enzyme is located on a plasmid in the bacterial cell, and at least one gene encoding the at least one trimethylamine catabolism enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the at least one gene encoding the at least one trimethylamine catabolism enzyme is located in the chromosome of the bacterial cell, and at least one gene encoding the at least one trimethylamine catabolism enzyme from a different species of bacteria is located in the chromosome of the bacterial cell. For example, E. coli comprises native formaldehyde dehydrogenase and formate dehydrogenase genes.

[0207] In some embodiments, the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed on a low-copy plasmid. In some

embodiments, the at least one gene encoding the at least one trimethylamine catabolism enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the at least one trimethylamine catabolism enzyme, thereby increasing the catabolism of trimethylamine.

Transporter (Importer) of TMA and/or TMAQ

[0208] In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more transporters that facilitate the uptake of TMA and/or TMAO.

[0209] In some embodiments, the transporter is a trimethylamine permease. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more copies of Trimethylamine permease from Hyphomicrobium denitrificans. In some

embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more copies of Trimethylamine permease from Methanosarcina barkeri. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more copies of Trimethylamine permease from Methanosarcina mazei. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more copies of Trimethylamine permease from Methanolobus psychrophilus R15. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more copies of

Trimethylamine permease from Candidatus Methanomassiliicoccus intestinalis Issoire-Mxl . Other proteins that mediate the import of TMA are well known to those of skill in the art. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more copies of a trimethylamine permease selected from Trimethylamine permease from

Hyphomicrobium denitrificans, Trimethylamine permease from Methanosarcina barkeri, Trimethylamine permease from Methanosarcina mazei, Trimethylamine permease from Methanolobus psychrophilus R15, Trimethylamine permease from Candidatus

Methanomassiliicoccus intestinalis Issoire-Mxl , and combinations thereof.

[0210] In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more TMAO transporter(s). Several TMAO transporters or permeases are known in the art. In some embodiments, the TMAO transporter is a TMAO- specific ABC transporter found in a number of divergent marine bacteria, including MRC and SAR11 clade Alphaproteobacteria, SAR324 cl&deDeltaproteobacteria, and

some Gammaproteobacteria {e.g., as described by Lidbury et al., Trimethylamine N-oxide metabolism by abundant marine heterotrophic bacteria Proc Natl Acad Sci U S A. 2014 Feb 18; 111(7): 2710-2715). In some embodiments, the TMAO transporter is a TMAO-specific transporter from Methylocella silvestris (see e.g., Zhu et al., Environ Microbiol. 2014

Oct;16(10):3318-30).

[0211] TMA and/or TMAO transporters, e.g., TMA and/or TMAO importers, may be expressed or modified in the bacteria of the invention in order to enhance TMA transport into the cell. Specifically, when the importer of TMA and/or TMAO is expressed in the recombinant bacterial cells of the invention, the bacterial cells import more TMA and/or TMAO into the cell when the importer is present and/or expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the engineered bacteria comprising gene sequence(s) encoding one or more transporter(s) of TMA and/or TMAO may be used to import TMA and/or TMAO into the bacteria to provide additional substrate for trimethylamine catabolism enzyme(s) expressed in the organism, which can be used to treat disorders in which TMA and/or TMAO is detrimental. In one embodiment, the bacterial cell of the invention comprises a heterologous gene sequence(s) encoding a transporter (importer) of TMA and/or TMAO. In one embodiment, the bacterial cell of the invention comprises a heterologous gene sequence(s) encoding transporter of TMA and/or TMAO and one or more heterologous gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s). Thus, in some embodiments, the invention provides a bacterial cell that comprises one or more heterologous gene sequence(s) encoding a trimethylamine catabolism enzyme operably linked to a first promoter and one or more heterologous gene sequence(s) encoding a transporter (importer) of TMA and/or TMAO. In some embodiments, the one or more heterologous gene sequence(s) encoding a transporter (importer) of TMA and/or TMAO may also be operably linked to the first promoter. In another embodiment, the invention provides a bacterial cell that comprises one or more heterologous gene sequence(s) encoding one or more TMA and/or TMAO catabolism enzyme(s) operably linked to a first promoter and one or more heterologous gene sequence(s) encoding a transporter (importer) of TMA and/or TMAO operably linked to a second promoter. In one embodiment, the first promoter and the second promoter are separate copies of the same promoter. In another embodiment, the first promoter and the second promoter are different promoters.

[0212] In one embodiment, the bacterial cell comprises non-native gene sequence(s) encoding one or more transporter (s) (importer) of TMA and/or TMAO from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises native gene sequence encoding a transporter (importer) of TMA and/or TMAO. In some embodiments, the native gene sequence encoding a transporter (importer) of TMA and/or TMAO is not modified. In another embodiment, the bacterial cell comprises more than one copy of a native gene sequence(s) encoding a transporter (importer) of TMA and/or TMAO. In another embodiment, the bacterial cell comprises one or more native gene sequence(s) encoding a transporter (importer) of TMA and/or TMAO, and comprises one or more non- native gene sequence(s) encoding a transporter of TMA and/or TMAO from a different bacterial species. In some embodiments, the bacterial cell comprises one or more, e.g. , two, three, four, five, or six copies of the native gene sequence(s) encoding a transporter of TMA and/or TMAO and/or comprises one or more, e.g. , two, three, four, five, or six copies of the non-native gene sequence(s) encoding a transporter of TMA and/or TMAO. In one embodiment, the bacterial cell comprises multiple copies of one or more heterologous gene sequence(s) encoding a transporter of TMA and/or TMAO.

[0213] The present invention further comprises gene sequence encoding functional fragments of a transporter of TMA and/or TMAO or functional variants of a transporter of TMA and/or TMAO. As used herein, the term "functional fragment thereof or "functional variant thereof of a transporter of TMA and/or TMAO relates to an element having qualitative biological activity in common with the wild-type transporter of TMA and/or TMAO from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated transporter of TMA and/or TMAO protein is one which retains essentially the same ability to import TMA and/or TMAO into the bacterial cell as does the transporter protein from which the functional fragment or functional variant was derived. In one embodiment, the recombinant bacterial cell of the invention comprises one or more heterologous gene sequence(s) encoding a functional fragment of a transporter of TMA and/or TMAO. In another embodiment, the recombinant bacterial cell of the invention comprises one or more heterologous gene sequence(s) encoding a functional variant of a transporter of TMA and/or TMAO.

[0214] Assays for testing the activity of a transporter of, a transporter of TMA and/or TMAO functional variant, or a transporter of TMA and/or TMAO functional fragment are well known to one of ordinary skill in the art.

[0215] In one embodiment the gene sequence(s) encoding the one or more

transporter(s) of TMA and/or TMAO have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the transporter of TMA and/or TMAO have been codon-optimized for use in Escherichia coli.

[0216] The present invention also encompasses bacteria comprising gene sequence(s) encoding one or more transporter(s) of TMA and/or TMAO that contain amino acids in its sequence that are substantially the same as a transporter amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.

[0217] In some embodiments, the one or more gene sequence(s) encoding a transporter of TMA and/or TMAO is mutagenized; mutants exhibiting increased TMA and/or TMAO transport are selected; and the mutagenized one or more gene sequence(s) encoding a transporter of TMA and/or TMAO is isolated and inserted into the bacterial cell of the invention. In some embodiments, the gene sequence(s) encoding one or more

transporter(s)of TMA and/or TMAO is mutagenized; mutants exhibiting decreased TMA and/or TMAO transport are selected; and the mutagenized one or more gene sequence(s) encoding a transporter of TMA and/or TMAO is isolated and inserted into the bacterial cell of the invention. The transporter modifications described herein may be present on a plasmid or chromosome.

[0218] In some embodiments, the engineered bacteria comprise gene(s) or gene cassette(s) encoding one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) described herein. In one embodiment the one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) have at least about 80% identity with one or more of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. In another embodiment, the one or more Trimethylamine and/or trimethylamine N- oxide transporter polypeptide(s) have at least about 85% identity with one or more of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. In one embodiment, the one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) have at least about 90% identity with one or more of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. In one embodiment, the one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) have at least about 95% identity with one or more of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. In another embodiment, the one or more

Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. Accordingly, in one embodiment, the one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. In another embodiment, the one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) comprise a sequence selected from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64. In yet another embodiment, the one or more Trimethylamine and/or trimethylamine N-oxide transporter polypeptide(s) encoded by the TMA catabolism gene(s) or gene cassette(s) and expressed by the genetically engineered bacteria consist of a sequence selected from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64.

[0219] In some embodiments, the bacterial cell comprises gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s) operably linked to a first promoter and gene sequence(s) encoding one or more TMA and/or TMAO transporter(s). In some embodiments, the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) is operably linked to the same copy or a different copy of the same promoter as the gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s) (first promoter). In other embodiments, the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) is operably linked to a promoter that is different (second promoter) from the promoter linked to the gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s) (first promoter). In one embodiment, the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) is directly operably linked to the second promoter. In another embodiment, the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) is indirectly operably linked to the second promoter. Thus, in some

embodiments, the expression of the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) is controlled by a different promoter than the promoter that controls expression of the gene sequence(s) encoding the one or more trimethylamine catabolism enzyme(s). In some embodiments, the expression of the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) is controlled by the same promoter that controls expression of the gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s). In some embodiments, the gene sequence(s) encoding a TMA and/or TMAO transporter and the gene sequence(s) encoding the trimethylamine catabolism enzyme are divergently transcribed from a promoter region. In some embodiments, expression of each of the genes encoding the one or more TMA and/or TMAO transporter(s) and the gene(s) encoding the one or more trimethylamine catabolism enzyme(s) is controlled by different promoters.

[0220] In one embodiment, the gene sequence(s) encoding the one or more TMA and/or TMAO transporter(s) is operably linked to a promoter that is not its natural promoter. In some embodiments, the gene sequence(s) encoding the one or more TMA and/or TMAO transporter(s) is operably linked to its native promoter. In some embodiments, the gene sequence(s) encoding the one or more TMA and/or TMAO transporter(s) is operably linked to an inducible promoter, such as any of the inducible promoters disclosed herein. In some embodiments, the gene sequence(s) encoding the one or more TMA and/or TMAO transporter(s) is operably linked to a promoter that is stronger than its native promoter. In some embodiments, the gene sequence(s) encoding the one or more TMA and/or TMAO transporter(s) is operablylinked to a constitutive promoter.

[0221] In some embodiments, the gene sequence(s) encoding the one or more TMA and/or TMAO transporter(s) is located on a plasmid in the bacterial cell. In another embodiment, the gene sequence(s) encoding the one or more TMA and/or TMAO

transporter(s) is located in the chromosome of the bacterial cell. In other embodiments, the bacteria comprises a native copy of the gene sequence encoding the TMA and/or TMAO transporter, which gene sequence is located in the chromosome of the bacterial cell, and gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) from a different species of bacteria, which gene sequence(s) is located on a plasmid in the bacterial cell. In other embodiments, both the native copy of the gene sequence encoding the TMA and/or TMAO transporter and the gene sequence(s) encoding one or more TMA and/or TMAO

transporter(s) from a different species of bacteria are located on a plasmid in the bacterial cell. In yet other embodiments, both the native copy of the gene sequence encoding the TMA and/or TMAO transporter and the gene sequence(s) encoding one or more TMA and/or TMAO transporter(s) from a different species of bacteria are located in the chromosome of the bacterial cell.

[0222] In some embodiments, the at least one native gene encoding the transporter of TMA and/or TMAO in the bacterial cell is not modified, and one or more additional copies of the native transporter of TMA and/or TMAO are inserted into the genome. In one

embodiment, the one or more additional copies of the native transporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the gene sequence(s) encoding the one or more trimethylamine catabolism enzyme(s), e.g., the FNR responsive promoter. In alternate embodiments, the at least one native gene encoding the TMA and/or TMAO transporter is not modified, and one or more additional copies of a TMA and/or TMAO transporter from a different bacterial species is inserted into the genome of the bacterial cell. In one embodiment, the one or more additional copies of the TMA and/or TMAO transporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the one or more gene sequence(s) encoding the trimethylamine catabolism enzyme(s), e.g., the FNR responsive promoter.

[0223] In some embodiments, when the TMA and/or TMAO transporter is expressed in the bacterial cells, the bacterial cells import 10% more TMA and/or TMAO into the bacterial cell as compared with bacteria of the same bacterial subtype under the same conditions that do not express a TMA and/or TMAO transporter. In some embodiments, when the TMA and/or TMAO transporter is expressed in the recombinant bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more TMA and/or TMAO into the bacterial cell as compared with bacteria of the same bacterial subtype under the same conditions that do not express a TMA and/or TMAO transporter. In some mbodiments, when the TMA and/or TMAO transporter is expressed in the bacterial cells, the bacterial cells import two-fold more TMA and/or TMAO into the bacterial cell as compared with bacteria of the same bacterial subtype under the same conditions that do not express a TMA and/or TMAO transporter. In other embodiments, when the TMA and/or TMAO transporter is expressed in the recombinant bacterial cells, the bacterial cells import threefold, four-fold, five-fold, six- fold, seven- fold, eight-fold, nine-fold, or ten- fold more TMA and/or TMAO into the bacterial cell as compared with bacteria of the same bacterial subtype under the same conditions that do not express a TMA and/or TMAO transporter.

[0224] In some embodiments, the bacterial cell comprises a genetic mutation in one or more endogenous gene(s) encoding a transporter (importer) of TMA and/or TMAO, wherein the genetic mutation reduces influx of TMA and/or TMAO into the bacterial cell. Without wishing to be bound by theory, such mutations may decrease intracellular TMA and/or TMAO concentrations and increase the flux through TMA catabolism pathways

Inducible Promoters

[0225] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the at least one gene encoding a trimethylamine catabolism enzyme such that the trimethylamine catabolism enzyme 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. In some embodiments, bacterial cell comprises two or more distinct trimethylamine catabolism enzymes. In some embodiments, the genetically engineered bacteria comprise multiple copies of the same trimethylamine catabolism enzyme gene. In some embodiments, the genetically engineered bacteria comprise multiple copies of different trimethylamine catabolism enzyme genes. In some embodiments, the at least one gene encoding the trimethylamine catabolism enzyme is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene encoding the trimethylamine catabolism enzyme is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some

embodiments, the at least one gene encoding the trimethylamine catabolism enzyme is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene encoding the trimethylamine catabolism enzyme is present in the chromosome and operably linked to a promoter that is induced under low- oxygen or anaerobic conditions. In some embodiments, the at least one gene encoding the trimethylamine catabolism enzyme is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.

[0226] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the at least one gene encoding a transporter of

trimethylamine and/or trimethylamine-N-oxide, such that the transporter, 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. In some embodiments, bacterial cell comprises two or more distinct copies of the at least one gene encoding a trimethylamine and/or trimethylamine-N- oxide transporter. In some embodiments, the genetically engineered bacteria comprise multiple copies of the same at least one gene encoding a trimethylamine and/or

trimethylamine-N-oxide transporter. In some embodiments, the at least one gene encoding a transporter of trimethylamine and/or trimethylamine-N-oxide , is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, is present on a plasmid and operably linked to a promoter that is induced under inflammatory conditions. In some embodiments, the at least one gene encoding a transporter trimethylamine and/or trimethylamine-N-oxide is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose. In some embodiments, the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, is present in the chromosome and operably linked to a promoter that is induced under inflammatory conditions. In some embodiments, the at least one gene encoding a transporter trimethylamine and/or trimethylamine-N-oxide is present in the chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.

[0227] In some embodiments, the promoter that is operably linked to the gene encoding the trimethylamine catabolism enzyme and the promoter that is operably linked to the gene encoding the trimethylamine and/or trimethylamine-N-oxide transporter, is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding the trimethylamine and/or trimethylamine-N-oxide catabolism enzyme and the promoter that is operably linked to the gene encoding the trimethylamine and/or trimethylamine-N-oxide transporter, is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by inflammatory conditions that may be found in the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the bacterial cell.

[0228] In some embodiments, the promoter that is operably linked to the at least one gene encoding the at least one trimethylamine catabolism enzyme is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the at least one gene encoding the at least one trimethylamine catabolism enzyme is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by inflammatory conditions such as conditions that may be found in a mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is coadministered with the bacterial cell.

FNR Dependent Regulation

[0229] In certain embodiments, the bacterial cell comprises at least one gene encoding at least one trimethylamine catabolism enzyme is expressed under the control of the fumarate and nitrate reductase regulator (FNR) promoter. In certain embodiments, the bacterial cell comprises at least one gene encoding a trimethylamine transporter is expressed under the control of the fumarate and nitrate reductase regulator (FNR) promoter. In E. coli, FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et ah, 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive.

[0230] FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.

Table 2: FNR responsive promoters

Table 3: Additional FNR Promoter Sequences

CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTACAGCAAACGGTCTGTA CGCTGTCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGTCAGCATAA CACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCGTCCGGCCTTTTCC

nirB2 TCTCTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGC SEQ ID NO: 22 ACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATAT

ACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAAT CGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaaga aggagatatacat

GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCGTCC

nirB3 GGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTC

TATTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAA SEQ ID NO: 23 TCAGCAATATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGG

GTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA

ydfZ ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTCATGCATGC

ATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTA SEQ ID NO: 24 TATTGCGCCCGTTACGTGGGCTTCGACTGTAAATCAGAAAGGAGAAAACACCT

GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTATCGTCGTCC GGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTTGTC

nirB+RBS TGTTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAA SEQ ID NO: 25 TCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGG

GTTGCTGAATCGTTAAGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATA TACAT ydfZ+RBS CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTCATGCATG

CATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGTATTT SEQ ID NO: 26 ATATTGCGCCCGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTAACAAAA

fnrSl GCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAGTTTGAGCGAAGTCAATAAAC SEQ ID NO: 27 TCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTAACT

TTAAGAAGGAGATATACAT

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTAACAAAA

fnrS2 GCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGAAGTCAATAAAC SEQ ID NO: 28 TCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCAAAGTGAACTCTAGAAATAATTTT

GTTTAACTTTAAGAAGGAGATATACAT

TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGTCAGCATAACACCC TGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCGTCCGGCCTTTTCCTCTCT

nirB+crp TCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAA

CATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCA SEQ ID NO: 29 TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTA

AGGTAGaaatgtgatctagttcacatttGCGGTAATAGAAAAGAAATCGAGGCAAAAatg tttgtttaactttaagaaggagatatacat

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTAACAAAA

fnrS+crp GCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGAAGTCAATAAAC SEQ ID NO: 30 TCTCTACCCATTCAGGGCAATATCTCTCaaatgtgatctagttcacattttttgtttaac tttaagaaggagatatacat

[0231] In one embodiment, the FNR responsive promoter comprises SEQ ID NO: 4. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 5. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 6. In another

embodiment, the FNR responsive promoter comprises SEQ ID NO: 7. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 8. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 19. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 20. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 21. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 22. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 23. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 24. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 25. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 26. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 27. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 28. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 29. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 30.

[0232] In other embodiments, the FNR responsive promoter has at least about 80% identity with a nucleic acid sequence encoding any of SEQ ID NOs:4-8 and SEQ ID NOs: 19- 30. In other embodiments, the FNR responsive promoter has at least about 85% identity with a nucleic acid sequence encoding any of SEQ ID NOs:4-8 and SEQ ID NOs: 19-30. In other embodiments, the FNR responsive promoter has at least about 90% identity with a nucleic acid sequence encoding any of SEQ ID NOs:4-8 and SEQ ID NOs: 19-30. In other embodiments, the FNR responsive promoter has at least about 95% identity with a nucleic acid sequence encoding any of SEQ ID NOs:4-8 and SEQ ID NOs: 19-30. In other embodiments, the FNR responsive promoter has at least about 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding any of SEQ ID NOs:4-8 and SEQ ID NOs: 19- 30. Accordingly, in some embodiments, the FNR responsive promoter has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding any of SEQ ID NOs:4-8 and SEQ ID NOs: 19-30.

[0233] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise at least one gene encoding at least one trimethylamine catabolism enzyme disclosed herein which is expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997). In alternate embodiments, the genetically engineered bacteria comprise at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter which is expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al, 2010) or ANR (Ray et ah, 1997). In these embodiments, catabolism of trimethylamine and/or trimethylamine-N-oxide is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.

[0234] In some embodiments, the bacterial cell of the invention comprises an oxygen- level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter from a different bacterial species. The heterologous oxygen-level dependent transcriptional regulator and promoter increase the transcription of genes operably linked to said promoter, e.g., the at least one gene encoding the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N- oxide transporter, in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non- native oxygen- level dependent transcriptional regulator is an FNR protein from N.

gonorrhoeae (see, e.g., Isabella et ah, 2011). In some embodiments, the corresponding wild- type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.

[0235] In some embodiments, the genetically engineered bacteria comprise a wild- type oxygen- level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the at least one gene encoding the the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild- type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and

corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the at least one gene encoding the the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen- level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et ah, 2006).

[0236] In some embodiments, the bacterial cells disclosed herein comprise multiple copies of the endogenous gene encoding the oxygen level- sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the at least one gene encoding the at least one trimethylamine catabolism enzyme are present on different plasmids. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the at least one gene encoding the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine transporter are present on different plasmids. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the at least one gene encoding the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter are present on the same plasmid.

[0237] In some embodiments, the gene encoding the oxygen level- sensing

transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the at least one gene encoding the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the at least one gene encoding the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or

trimethylamine-N-oxide transporter are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the at least one gene encoding the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the at least one trimethylamine catabolism enzyme and/or the at least one gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter. In some embodiments, the transcriptional regulator and the at least one trimethylamine catabolism enzyme are divergently transcribed from a promoter region.

[0238] In some embodiments, the expression of the at least one trimethylamine catabolism gene decreases the levels of one or more trimethylaminuria biomarkers. In some embodiments, the at least one trimethylamine catabolism gene expressed by the genetically engineered bacteria decreases the levels of one or more trimethylaminuria biomarkers.

RNS dependent regulation

[0239] In some embodiments, the genetically engineered bacteria comprise at least one gene encoding at least one trimethylamine catabolism enzyme that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium that expresses a trimethylamine catabolism enzyme and/or a transporter of trimethylamine is under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the trimethylamine catabolism enzyme and/or a transporter of trimethylamine is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.

[0240] As used herein, "reactive nitrogen species" and "RNS" are used

interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO*), peroxynitrite or peroxynitrite anion (ONOO-), nitrogen dioxide (·Ν02), dinitrogen trioxide (N203), peroxynitrous acid

(ONOOH), and nitroperoxycarbonate (ONOOC02-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

[0241] As used herein, "RNS-inducible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g. , a trimethylamine catabolism enzyme gene sequence(s), e.g., any of the TMA and/or TMAO catabolism enzymes described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

[0242] As used herein, "RNS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., trimethylamine catabolism enzyme gene sequence(s), trimethylamine transporter sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

[0243] As used herein, "RNS-repressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS- repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

[0244] As used herein, a "RNS -responsive regulatory region" refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 4.

Table 4. Examples of RNS-sensing transcription factors and RNS-responsive genes

[0245] In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of an trimethylamine and/or trimethylamine-N-oxide catabolism enzyme, and/or trimethylamine and/or trimethylamine-N-oxide transporter, thus controlling expression of the trimethylamine and/or TMAO catabolism enzyme and/or trimethylamine and/or

trimethylamine-N-oxide transporter relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is an trimethylamine and/or trimethylamine-N-oxide catabolism enzyme and/or trimethylamine and/or

trimethylamine-N-oxide transporter, such as any of the trimethylamine and/or

trimethylamine-N-oxide catabolism enzymes and trimethylamine and/or trimethylamine-N- oxide transporters provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS- sensing transcription factor binds to and/or activates the regulatory region and drives expression of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the trimethylamine catabolism enzyme and/or transporter is decreased or eliminated.

[0246] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0247] 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 genes, e.g., one or more trimethylamine catabolism enzyme, and transporter gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the catabolism enzyme, transporter, and/or binding protein.

[0248] 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 gene or genes and producing one or more TMA and/or TMAO catabolism enzymes. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

[0249] 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.

[0250] 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 ah, 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 ah, 2009; Dunn et ah, 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 genes, e.g., atrimethylamine catabolism enzyme gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked trimethylamine catabolism enzyme and/or trimethylamine transporter gene or genes and producing the encoding a TMA and/or TMAO catabolism enzyme(s).

[0251] 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.

[0252] 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.

[0253] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a TMA and/or TMAO catabolism enzyme. The two repressor activation regulatory circuit comprises a first RNS- sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., encoding a TMA and/or TMAO catabolism enzyme. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene or genes is expressed.

[0254] 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).

[0255] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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 trimethylamine catabolism enzyme 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 trimethylamine catabolism enzyme 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 trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter in the presence of RNS.

[0260] In some embodiments, the gene or gene cassette for producing the anti- inflammation and/or gut barrier function enhancer molecule is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0261] In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the TMA and/or TMAO catabolism enzyme(s) and also permits fine- tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to 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.

ROS-dependent regulation

[0262] In some embodiments, the genetically engineered bacteria comprise a gene for producing a trimethylamine catabolism enzyme and/or trimethylamine and/or

trimethylamine-N-oxide transporter that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium that expresses a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the trimethylamine catabolism enzyme is expressed under the control of a cellular damaged- dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.

[0263] As used herein, "reactive oxygen species" and "ROS" are used

interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal- catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H202), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical (·ΟΗ), superoxide or superoxide anion (·02-), singlet oxygen (102), ozone (03), carbonate radical, peroxide or peroxyl radical (·02-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOCl), nitric oxide (NO*), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et ah, 2014).

[0264] As used herein, "ROS-inducible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g. , a sequence or sequences encoding one or more TMA and/or TMAO catabolism enzyme(s). For example, in the presence of ROS, a transcription factor, e.g. , OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

[0265] As used herein, "ROS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more TMAO and/or TMAO catabolism enzyme(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

[0266] As used herein, "ROS-repressible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS- repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

[0267] As used herein, a "ROS -responsive regulatory region" refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 5.

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

[0268] In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a TMA and/or TMAO catabolism enzyme, thus controlling expression of the trimethylamine catabolism enzyme relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is an TMA and/or TMAO catabolism enzyme; when ROS is present, e.g. , in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter thereby producing the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter.

Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter is decreased or eliminated.

[0269] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS ; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0270] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR "functions primarily as a global regulator of the peroxide stress response" and is capable of regulating dozens of genes, e.g. , "genes involved in H202 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)" and "OxyS, a small regulatory RNA" (Dubbs et ah , 2012). The genetically engineered bacteria may comprise any suitable ROS-responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g. , Zheng et ah , 2001 ; Dubbs et ah , 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g. , a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene. In the presence of ROS, e.g., H202, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene and producing the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROS-inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.

[0271] In alternate embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is "activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression" (Koo et al, 2003). "SoxR is known to respond primarily to superoxide and nitric oxide" (Koo et al., 2003), and is also capable of responding to H202. The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al, 2003; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., a TMA and/or TMAO catabolism enzyme. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene and producing an TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter.

[0272] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0273] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR "binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event," but oxidized OhrR is "unable to bind its DNA target" (Duarte et al, 2010). OhrR is a "transcriptional repressor [that] ... senses both organic peroxides and NaOCl" (Dubbs et al., 2012) and is "weakly activated by H202 but it shows much higher reactivity for organic hydroperoxides" (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g. , a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene. In the presence of ROS, e.g., NaOCl, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked

trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene and producing the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter.

[0274] OhrR is a member of the MarR family of ROS -responsive regulators. "Most members of the MarR family are transcriptional repressors and often bind to the - 10 or -35 region in the promoter causing a steric inhibition of RNA polymerase binding" (Bussmann et ah , 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et ah , 2012).

[0275] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is "a MarR-type transcriptional regulator" that binds to an "18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA" and is "reversibly inhibited by the oxidant H202" (Bussmann et ah , 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to "a putative polyisoprenoid-binding protein (cgl322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cgl426), two putative FMN reductases (cgl l50 and cgl850), and four putative monooxygenases (cg0823, cgl848, cg2329, and cg3084)" (Bussmann et ah , 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g. , Bussmann et ah , 2010; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g. , an TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter. In the presence of ROS, e.g., H202, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter and producing the TMA and/or TMAO

catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter.

[0276] In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[0277] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0278] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR "when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)" (Marinho et al, 2014). PerR is a "global regulator that responds primarily to H202" (Dubbs et ah, 2012) and "interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes" (Marinho et al, 2014). PerR is capable of binding a regulatory region that "overlaps part of the promoter or is immediately downstream from it" (Dubbs et ah, 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al, 2012; Table 1).

[0279] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a TMA and/or TMAO catabolism enzyme. The two repressor activation regulatory circuit comprises a first ROS- sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a TMA and/or TMAO catabolism enzyme. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a TMA and/or TMAO catabolism enzyme. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a TMA and/or TMAO catabolism enzyme. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter is expressed.

[0280] A ROS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although "OxyR is primarily thought of as a transcriptional activator under oxidizing conditions... OxyR can function as either a repressor or activator under both oxidizing and reducing conditions" (Dubbs et ah, 2012), and OxyR "has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)" (Zheng et ah, 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et ah, 2001; Table 1). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by RosR. In addition, "PerR- mediated positive regulation has also been observed...and appears to involve PerR binding to distant upstream sites" (Dubbs et al, 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by PerR.

[0281] One or more types of ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, "OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both" (Dubbs et ah, 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS -responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.

[0282] Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 6. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 31, 32, 33, or 34, or a functional fragment thereof.

Table 6. Nucleotide sequences of exemplary OxyR-regulated regulatory regions

Regulatory

01234567890123456789012345678901234567890123456789

sequence

TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACG

dps

CTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCG

(SEQ ID NO:

GAACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGA

32)

CATAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGA GAAAGGTACC

GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC

CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG

CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA

ahpC

CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC

(SEQ ID NO:

AAAGTTCACAAAGTTGTCTTACGAAGGTTGTAAGGTAAAACTT

33)

ATCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAAAT

TGGTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATG CGAATTCATTAAAGAGGAGAAAGGTACC

CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGC

oxyS

GATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTC

(SEQ ID NO:

TGACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGT

34)

ACC

[0283] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0284] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0285] In some embodiments, the genetically engineered bacteria comprise a ROS- sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from

Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROS- sensing transcription factor, e.g., OxyR, is deleted or mutated to reduce or eliminate wild- type activity.

[0286] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS- sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

[0287] In some embodiments, the genetically engineered bacteria comprise a wild- type gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a

corresponding regulatory region, e.g., a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the catabolism enzyme, transporter, and/or binding protein in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS -responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N- oxide transporter in the presence of ROS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the trimethylamine catabolism enzyme in the presence of ROS.

[0288] In some embodiments, the gene or gene cassette for producing the

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

[0289] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing an TMA and/or TMAO catabolism enzyme(s) and/or trimethylamine and/or trimethylamine-N-oxide transporter(s). In some embodiments, the gene(s) capable of producing a TMA and/or TMAO catabolism enzyme(s) and/or trimethylamine and/or trimethylamine-N-oxide transporter(s) is present on a plasmid and operatively linked to a ROS-responsive regulatory region. In some embodiments, the gene(s) capable of producing a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter is present in a chromosome and operatively linked to a ROS-responsive regulatory region.

[0290] Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more TMA and/or TMAO catabolism enzymes under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor.

[0291] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing an trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter such that the trimethylamine catabolism enzyme and/or transporter can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g. , in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N- oxide transporter. In some embodiments, the gene encoding the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the trimethylamine catabolism enzyme and/or trimethylamine and/or

trimethylamine-N-oxide transporter is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter. In some embodiments, the gene encoding the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter is expressed on a chromosome.

[0292] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MO As), e.g. , circuits producing multiple copies of the same product (e.g. , to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene encoding a particular trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene encoding a particular trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter inserted at three different insertion sites and three copies of the gene encoding a different trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N- oxide transporter inserted at three different insertion sites.

[0293] In some embodiments, under conditions where the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30- fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300- fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900- fold, at least about 1,000-fold, or at least about 1,500-fold more of the TMA and/or TMAO catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

[0294] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s). Primers specific for trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain trimethylamine catabolism enzyme mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s).

[0295] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s). Primers specific for trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93- 97° C, 55-65° C, and 35-45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter gene(s).

[0296] In other embodiments, the inducible promoter is a trimethylamine and/or trimethylamine-N-oxide responsive promoter.

Essential Genes and Auxotrophs

[0297] As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et ah, Essential genes on metabolic maps, Curr. Opin. BiotechnoL, 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).

[0298] 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 engineered bacteria of the disclosure becoming an auxotroph, e.g., the bacteria may be an auxotroph depending on the environmental conditions (a conditional 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.

[0299] 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 an oligonucleotide synthesis gene, for example, thy A. 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 ox Met A. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil, as long as the corresponding wild-type gene product is not produced in the bacteria.

[0300] Table 7 lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.

Table 7. Non-limiting Examples of Bacterial Genes Useful for Generation of an

Auxotroph

[0301] Table 8 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli. Table 8. Survival of amino acid auxotrophs in the mouse

[0302] 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 ah, 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).

[0303] 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 ah, 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).

[0304] 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 ah, 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).

[0305] 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 comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

[0306] Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, ML, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, UgA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc ftsB, eno, pyrG, chpR, Igt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, IpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, IpxA, IpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, Int, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, IpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabl, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

[0307] 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).

[0308] In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some

embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G.

[0309] 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, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are

complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2- aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

[0310] 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).

[0311] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.

[0312] 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 engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). 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).

Genetic Regulatory Circuits

[0313] In some embodiments, the genetically engineered bacteria comprise multi- layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811, incorporated herein by reference in its entirety). The genetic regulatory circuits are useful to screen for mutant bacteria that produce a trimethylamine catabolism enzyme and/or trimethylamine and/or trimethylamine-N-oxide transporter or rescue an auxotroph. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.

[0314] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the payload is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the payload is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

[0315] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the tetR, and the payload is expressed.

[0316] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.

[0317] Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

[0318] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR- responsive promoter, and a second gene or gene cassette for producing a payload. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the payload. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing payload translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.

[0319] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed.

[0320] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a payload operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR- responsive promoter, the recombinase is not expressed, the payload remains in the 3' to 5' orientation, and no functional payload is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the payload is reverted to the 5' to 3' orientation, and functional payload is produced.

[0321] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload. The third gene encoding the T7 polymerase is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3' to 5' orientation, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5' to 3' orientation, and the payload is expressed.

Kill Switches

[0322] In some embodiments, the genetically engineered bacteria also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329, each of which are expressly 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.

[0323] 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, genes or gene cassette(s), for example, a therapeutic gene(s) or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following expression of the catabolism enzyme cassette(s) and/or gene(s) present in the engineered bacteria. In some embodiments, the kill switch is activated in a delayed fashion following expression of the heterologous gene(s) or gene cassette(s), for example, after the production of the

corresponding protein(s) or molecule(s). Alternatively, the bacteria may be engineered to die after the bacteria has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject).

[0324] 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 β-D-l-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et ah, 2010). In some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of a heterologous gene(s) or gene cassette(s). In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of a heterologous gene(s) or gene cassette(s).

[0325] 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; i.e., an activation-based kill switch) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased {i.e., a repression-based kill switch).

[0326] 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 leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In 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 engineered bacterial cell is no longer viable.

[0327] 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.

[0328] 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.

[0329] 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. Accordingly, in one embodiment, the disclosure provides at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 recombinases that can be used serially.

[0330] 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 engineered bacterial cell is not viable after the first essential gene is excised.

[0331] 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.

[0332] In one embodiment, the first excision enzyme is Xisl. In one embodiment, the first excision enzyme is Xis2. In one embodiment, the first excision enzyme is Xisl, and the second excision enzyme is Xis2. [0333] 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.

[0334] In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxbl, PhiC31, TP901, Bxbl, PhiC31, TP901, HK022, HPl, R4, Intl, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, IntlO, Intl l, Intl2, Intl3, Intl4, Intl5, Intl6, Intl7, Intl8, Intl9, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.

[0335] 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. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) are described herein. The disclosure provides engineered bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the engineered bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the arciBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the arciBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the arciBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxin gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arabinose system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

[0336] 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 arciBAD 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.

[0337] Arabinose inducible promoters are known in the art, including Para, ParaB, Parac, and ParaBAD- In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the Parac promoter and the ParaBAD promoter operate as a bidirectional promoter, with the ParaBAD promoter controlling expression of a heterologous gene(s) in one direction, and the Parac (in close proximity to, and on the opposite strand from the ParaBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

[0338] In one exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a Parac promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (PTetiO- In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the engineered bacterial cell, and the engineered 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.

[0339] In one embodiment of the disclosure, the engineered 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 engineered bacterial cell. The engineered 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 engineered bacterial cell will be killed by the toxin.

[0340] In another embodiment of the disclosure, the engineered bacterial cell further comprises an antitoxin under the control of the ParaBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the engineered bacterial cell. The engineered bacterial cell is no longer viable once the toxin protein is expressed, and the engineered bacterial cell will be killed by the toxin.

[0341] 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 ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell (and required for survival), and a Parac promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the engineered bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and the essential protein required for survival is not expressed. In this case, the engineered bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anto-toxin kill- switch system described directly above.

[0342] In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer- lived toxin killing it. [0343] In some embodiments, the engineered bacteria of the present disclosure, for example, bacteria described herein may further comprise the gene(s) encoding the

components of any of the above-described kill-switch circuits.

[0344] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, lbs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hip A, microcin B, microcin B 17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin lb, colicin 5, colicinlO, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

[0345] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNAl, yafQ, CcdA, MazE, ParD, yaf , Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccE^CTD, MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, ImlO, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

[0346] 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.

[0347] In one embodiment, the method further comprises administering a second engineered bacterial cell to the subject, wherein the second engineered bacterial cell comprises a heterologous reporter gene operably linked to an inducible promoter that is directly or indirectly induced by an exogenous environmental condition. In one embodiment, the heterologous reporter gene is a fluorescence gene. In one embodiment, the fluorescence gene encodes a green fluorescence protein (GFP). In another embodiment, the method further comprises administering a second engineered bacterial cell to the subject, wherein the second engineered bacterial cell expresses a lacZ reporter construct that cleaves a substrate to produce a small molecule that can be detected in urine (see, for example, Danio et ah, Science Translational Medicine, 7(289): 1-12, 2015, the entire contents of which are expressly incorporated herein by reference). Isolated Plasmids

[0348] In other embodiments, the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a first payload operably linked to a first inducible promoter, and a second nucleic acid encoding a second payload operably linked to a second inducible promoter. In other embodiments, the disclosure provides an isolated plasmid further comprising a third nucleic acid encoding a third payload operably linked to a third inducible promoter. In other embodiments, the disclosure provides a plasmid comprising four, five, six, or more nucleic acids encoding four, five, six, or more payloads operably linked to inducible promoters. In any of the embodiments described here, the first, second, third, fourth, fifth, sixth, etc "payload(s)" can be a trimethylamine catabolism enzyme, a trimethylamine and/or trimethylamine-N-oxide transporter, or other sequence described herein. In one embodiment, the nucleic acid encoding the first payload and the nucleic acid encoding the second payload are operably linked to the first inducible promoter. In one embodiment, the nucleic acid encoding the first payload is operably linked to a first inducible promoter and the nucleic acid encoding the second payload is operably linked to a second inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In another embodiment, the first inducible promoter and the second inducible promoter are different inducible promoters. In other embodiments comprising a third nucleic acid, the nucleic acid encoding the third payload and the nucleic acid encoding the first and second payloads are all operably linked to the same inducible promoter. In other embodiments, the nucleic acid encoding the first payload is operably linked to a first inducible promoter, the nucleic acid encoding the second payload is operably linked to a second inducible promoter, and the nucleic acid encoding te third payload is operably linked to a third inducible promoter. In some embodiments, the first, second, and third inducible promoters are separate copies of the same inducible promoter. In other embodiments, the first inducible promoter, the second inducible promoter, and the third inducible promoter are different inducible promoters. In some embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter, are each directly or indirectly induced by low-oxygen or anaerobic conditions. In other embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter, are each a fumarate and nitrate reduction regulator (FNR) responsive promoter. In other embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter are each a ROS-inducible regulatory region. In other embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter are each a RNS-inducible regulatory region.

[0349] In some embodiments, the at least one heterologous gene encoding a trimethylamine catabolism enzyme is operably linked to a constitutive promoter. In one embodiment, the constitutive promoter is a lac promoter. In another embodiment, the constitutive promoter is a tet promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli σ32 promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli σ70 promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis, σΑ promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis σΒ promoter. In another embodiment, the constitutive promoter is a Salmonella promoter. In other embodiments, the constitutive promoter is a bacteriophage T7 promoter. In other embodiments, the constitutive promoter is and a bacteriophage SP6 promoter. In any of the above-described embodiments, the plasmid further comprises a heterologous gene encoding a trimethylamine and/or trimethylamine-N-oxide transporter, and/or a kill switch construct, which may be operably linked to a constitutive promoter or an inducible promoter.

[0350] In some embodiments, the isolated plasmid comprises at least one

heterologous catabolism enzyme gene operably linked to a first inducible promoter; a heterologous gene encoding a TetR protein operably linked to a ParaBAD promoter, a heterologous gene encoding AraC operably linked to a ParaC promoter, a heterologous gene encoding an antitoxin operably linked to a constitutive promoter, and a heterologous gene encoding a toxin operably linked to a PTetR promoter. In another embodiment, the isolated plasmid comprises at least one heterologous gene encoding a catabolism enzyme operably linked to a first inducible promoter; a heterologous gene encoding a TetR protein and an antitoxin operably linked to a ParaBAD promoter, a heterologous gene encoding AraC operably linked to a ParaC promoter, and a heterologous gene encoding a toxin operably linked to a PTetR promoter.

[0351] In one embodiment, the plasmid is a high-copy plasmid. In another embodiment, the plasmid is a low-copy plasmid.

[0352] In another embodiment, the bacterial cell further comprises a genetic mutation which reduces export of trimethylamine and/or trimethylamine-N-oxide from the bacterial cell. [0353] In another aspect, the disclosure provides a recombinant bacterial cell comprising an isolated plasmid described herein. In another embodiment, the disclosure provides a pharmaceutical composition comprising the recombinant bacterial cell.

Multiple Mechanisms of Action

[0354] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MO As), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dap A, cea, and other shown in Fig. 5. For example, the genetically engineered bacteria may include four copies of a trimethylamine catabolism gene or trimethylamine catabolism gene cassette, or four copies of a trimethylaminecatabolism gene inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include one or more copies of a trimethylamine catabolism gene or gene cassette inserted at one or more different insertion sites, e.g., malE/K, insB/I, and lacZ, one or more copies of a trimethylamine catabolism gene or gene cassette inserted at one or more different insertion sites, e.g., dapA, cea, and araC/BAD and/or one or more copies of a trimethylamine catabolism gene or gene cassette inserted at one or more different insertion sites.

[0355] In some embodiments, the genetically engineered bacteria comprise one or more of: one or more gene(s) and/or gene cassettes encoding one or more trimethylamine catabolism enzyme(s), in wild type or in a mutated form (for increased stability or metabolic activity); (2) one or more gene(s) and/or gene cassette(s) encoding one or more transporter(s) for uptake of trimethylamine and/or TMAO, in wild type or in mutated form (for increased stability or metabolic activity); (3) one or more gene(s) or gene cassette(s) encoding one or more trimethylamine catabolism enzyme(s) for secretion and extracellular degradation of trimethylamine and/or trimethylamine-N-oxide, (4) one or more gene(s) or gene cassette(s) encoding one or more components of secretion machinery, as described herein (5) one or more auxotrophies, e.g., deltaThyA; (6) one or more gene(s) or gene cassette(s) encoding one or more antibiotic resistance(s), including but not limited to, kanamycin or chloramphenicol resistance; (7) mutations/deletions in genes, as described herein; (10) mutations/deletions in genes of the endogenous trimethylamine and/or trimethylamine-N-oxide synthesis pathway.

[0356] In some embodiments, the genetically engineered bacteria comprise two or more different pathway cassettes or operons comprising trimethylamine catabolism enzymes. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding one or more trimethylamine catabolism enzymes.

[0357] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding one or more trimethylamine catabolism enzymes and one or more gene(s) or gene cassette(s) encoding one or more trimethylamine and/or trimethylamine-N-oxide transporters (importers), such as any of the trimethylamine and/or trimethylamine-N-oxide transporters described herein and otherwise known in the art.

[0358] In some embodiments, certain catalytic steps are rate limiting and in such a case it may be beneficial to add additional copies of one or more gene(s) encoding one or more rate limiting enzyme(s).

[0359] In some embodiments, each gene from a trimethylamine catabolism pathway described herein can be expressed individually, each under control of a separate (same or different) promoter.

[0360] In certain embodiments the order of the genes within a gene cassette can be modified, e.g., to increase or decrease levels of a particular gene within a cassette.

[0361] In any of the embodiments described in this section or elsewhere in the specification, any one or more the genes can be operably linked to a directly or indirectly inducible promoter, such as any of the promoters described herein, e.g., induced by low oxygen or anaerobic conditions, such as those found in the mammalian gut.

[0362] In certain embodiments, ribosome binding sites, e.g., stronger or weaker ribosome binding sites can be used to modulate (increase or decrease) the levels of expression of a catabolism enzyme within a cassette.

[0363] In some embodiments, the genetically engineered bacteria further comprise mutations or deletions, e.g., in an auxotrophy.

Host-Plasmid Mutual Dependency

[0364] In some embodiments, the genetically engineered bacteria also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et ah, 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad- spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wild- type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least one- hundred generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria described herein.

[0365] The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxo trophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

[0366] In some embodiments, the vector comprises a conditional origin of replication. In some embodiments, the conditional origin of replication is a R6K or ColE2- P9. In embodiments where the plasmid comprises the conditional origin of replication R6K, the host cell expresses the replication initiator protein π. In embodiments where the plasmid comprises the conditional origin or replication ColE2, the host cell expresses the replication initiator protein RepA. It is understood by those of skill in the art that the expression of the replication initiator protein may be regulated so that a desired expression level of the protein is achieved in the host cell to thereby control the replication of the plasmid. For example, in some embodiments, the expression of the gene encoding the replication initiator protein may be placed under the control of a strong, moderate, or weak promoter to regulate the expression of the protein.

[0367] In some embodiments, the vector comprises a gene encoding a protein required for complementation of a host cell auxotrophy, preferably a rich-media compatible auxotrophy. In some embodiments, the host cell is auxotrophic for thymidine (AthyA), and the vector comprises the thymidylate synthase (thyA) gene. In some embodiments, the host cell is auxotrophic for diaminopimelic acid (AdapA) and the vector comprises the 4-hydroxy- tetrahydrodipicolinate synthase (dapA) gene. It is understood by those of skill in the art that the expression of the gene encoding a protein required for complementation of the host cell auxotrophy may be regulated so that a desired expression level of the protein is achieved in the host cell. [0368] In some embodiments, the vector comprises a toxin gene. In some

embodiments, the host cell comprises an anti-toxin gene encoding and/or required for the expression of an anti-toxin. In some embodiments, the toxin is Zeta and the anti-toxin is Epsilon. In some embodiments, the toxin is Kid, and the anti-toxin is Kis. In preferred embodiments, the toxin is bacteriostatic. Any of the toxin/antitoxin pairs described herein may be used in the vector systems of the present disclosure. It is understood by those of skill in the art that the expression of the gene encoding the toxin may be regulated using art known methods to prevent the expression levels of the toxin from being deleterious to a host cell that expresses the anti-toxin. For example, in some embodiments, the gene encoding the toxin may be regulated by a moderate promoter. In other embodiments, the gene encoding the toxin may be cloned adjacent to ribosomal binding site of interest to regulate the expression of the gene at desired levels (see, e.g., Wright et al. (2015)).

Integration

[0369] 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. One or more copies of the heterologous gene or heterologous gene cassette may be integrated into the bacterial chromosome. Having multiple copies of the gene or gene cassette integrated into the chromosome allows for greater production of the corresponding protein(s) 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.

[0370] For example, Fig. 5 depicts a map of integration sites within the E. coli Nissle chromosome. Fig. 6 depicts three bacterial strains wherein the RFP gene has been successfully integrated into the bacterial chromosome at an integration site.

Secretion

[0371] In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism {e.g., gram positive bacteria) or non-native secretion mechanism {e.g., gram negative bacteria) that is capable of secreting the trimethylamine catabolism enzyme 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 (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.

[0372] 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 (TISS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance- nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al, 2015; Costa et al, 2015; Reeves et al, 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in the figures. 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).

[0373] In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type Ill-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. Thus, the molecule is secreted into the gut lumen or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the trimethylamine catabolism enzyme from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologouse protein or peptide, e.g., a trimethylamine catabolism enzyme, comprises a type III secretion sequence that allows the trimethylamine catabolism enzyme to be secreted from the bacteria. [0374] In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest, e.g., a trimethylamine catabolism enzyme. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[0375] In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in Fig. 9, a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex ('Beta-barrel assembly machinery') where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane-associated peptidase (black scissors; right side of Bam complex) to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologouse protein or peptide, e.g., a trimethylamine catabolism enzyme, comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.

[0376] In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. Fig. 10 shows the alpha- hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C- terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[0377] 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 ah , 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 catabolism enzymefrom the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.

[0378] In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g., therapeutic polypeptides) - particularly those of eukaryotic origin - contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

[0379] One way to secrete properly folded proteins in gram-negative bacteria- particularly those requiring disulphide bonds - is to target the periplasm in bacteria with a destabilized outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These "leaky" gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a "leaky" or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or

mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at -500,000 copies per cell and functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are deactived. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpl can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some

embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[0380] To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from lpp, ompA, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl, under the control of an inducible promoterFor example, expression of lpp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., ove expression of colicins or the third topological domain of TolA, which peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

[0381] Table 9: The tables below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

Table 9. Secretion systems for gram positive bacteria

Table 10. Secretion Systems for Gram negative bacteria

Tat (IISP) Twin- 2.A.6 + + + 2-4 PMF arginine 4 (chloroplas

targeting ts)

translocase

Oxal Cytochrome 2.A.9 + + + 1 None or (YidC) oxidase (mitochon PMF biogenesis dria

family chloroplast

s)

MscL Large 1.A.2 + + + 1 None conductance 2

mechanosens

itive channel

family

Holins Holin 1.E.1 + 1 None functional •21

superfamily

Eukaryotic Organelles

MPT Mitochondria 3.A. + >20 ATP

1 protein B (mitochon

translocase drial)

CEPT Chloroplast 3.A.9 (+) + >3 GTP envelope (chloroplas

protein ts)

translocase

Bcl-2 Eukaryotic 1.A.2 + 1? None

Bcl-2 family 1

(programmed

cell death)

Gram-negative bacterial outer membrane channel-forming translocases

MTB Main 3.A.1 +^b - 14 ATP; (IISP) terminal 5 PMF branch of the

general

secretory

translocase

FUP AT-1 Fimbrial 1.B.1 +^b None usher protein 1 +^b - None

^!

Autotransport 1.B.1

er-1 2

AT-2 Autotransport 1.B.4 +^b None OMF er-2 0 +^b + None

^!

(ISP) 1.B.1

7

TPS 1.B.2 + + None Secretin 0 +^b None

^!

(IISP and 1.B.2

IISP) 2 OmpIP Outer 1.B.3 + - + >4 None?

membrane 3 (mitochon

insertion dria;

porin chloroplast

s)

[0382] The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other trimethylamine catabolism enzyme from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe / Volume 1, Number 9, 2006 "Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently", the contents of which is herein incorporated by reference in its entirety.

[0383] In some embodiments, one or more trimethylamine and or trimethylamine N- oxide catabolic enzymes described herein are expressed by the genetically engineered bacteria in combination with any of the secretion systems described herein and are secreted. In some embodiments, the one or more trimethylamine and/or trimethylamine N-oxide catabolic enzymes described herein are further modified to improve secretion efficiency, decreased susceptibility to proteases, stability, and/or half-life.

[0384] Alternatively, any of the enzymes expressed by the genes described herein, e.g., in Fig. 1A, Fig. IB, and Fig. 2, may be combined.

In Vivo Models

[0385] The recombinant bacteria of the disclosure may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition in which

trimethylamine and/or trimethylamine-N-oxide is detrimental may be used. For example, a mouse may be fed with choline, a precursor of TMA, and plasma levels of TMA/TMAO can be monitored, as well as observation of fibrosis in the kidneys and kidney structure. The recombinant bacterial cells of the invention may be administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by measuring urine levels and/or plasma levels of trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) both before and after treatment. The animal may be sacrificed, and tissue samples may be collected and analyzed. Animal models are also known in the art. For example, an atherosclerosis mouse model as described by Gregory et al. can be used (see, e.g., Gregory et ah, 2015, . Biol. Chem., 290(9):5647-5660). [0386] Models of TMAO induced atherosclerosis and/or kidney disease are described in in Koeth et al., Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis; Nat Med. 2013 May; 19(5): 576-585 and Tang et al., Gut

Microbiota-Dependent Trimethylamine N-oxide (TMAO) Pathway Contributes to Both Development of Renal Insufficiency and Mortality Risk in Chronic Kidney Disease, Circ Res. 2015 Jan 30; 116(3): 448-455). Koeth et al. compared C57BL/6J, Apoe ^1' mice on normal chow diet versus a diet supplemented in L-carnitine for 15 weeks. The production of both d3-(methyl)TMA and d3-(methyl)TMAO following gastric gavage of d3- (methyl)carnitine was induced by approximately ten-fold in mice on the L-carnitine supplemented diet. Cholesterol catabolism is altered, including decreased reverse cholesterol transport and reduced bile acid pools, in the carnitine fed mice, which also display

accelerated atherosclerosis. Tang et al., compared C57BL6J mice fed with standard chow diet with a diet supplemented with choline (1.0% total) and with the same diet supplemented with TMAO (0.12%) and reported increased phosphorylation of Smad3, an important regulator of the pro-fibrotic TGF-p/Smad3 signaling pathway during fibrotic kidney disease and increased kidney injury marker- 1 (KIM-1) levels, cystatin C levels, along with renal histopatho logical and functional impairment in the mice fed the cholin and TMAO supplemented diets. In some embodiments, such models and feeding regimens can be used to assess the effect of providing the genetically engineered bacteria on TMA/TMAO levels and in the prevention or reduction of arthero sclerosis and/or chronic kidney disease.

[0387] In some embodiments, the efficacy of the genetically engineered bacteria or the extent of the disease is assessed using one or more biomarkers for chronic kidney disease as described herein or known in the art (by administering the engineered bacteria disclosed herein to an animal or cell system and measuring the level of biomarker, as compared with the administration of bacteria that do not express a TMA and/or TMAO catabolic enzyme). Chronic kidney disease (CKD) diagnosis, evaluation and treatment is based mainly on biomarkers that assess kidney function and can often be detected with non-invasive testing, and which are well known in the art (see e.g., Lopez- Giaco man and Madero, Biomarkers in chronic kidney disease, from kidney function to kidney damage World J Nephrol. 2015 Feb 6; 4(1): 57-73). Glomerular filtration rate (GFR) is a frequently used marker of kidney function. GFR is usually estimated from equations that take into account endogenous filtration markers like serum creatinine (SCr) and cystatin C (CysC). Blood urea nitrogen can also be used as an indicator of GFR. B2-microglobulin (B2-M) is elevated in kidney disease and can be used as a biomarker alone or in combination with GFR estimations. Albumin excretion rate (AER) can be determined in 24 h urine collections or in spot collections and can be used alone or in combination with GFR estimations. Combining albuminuria with eGFR improves the prediction of CKD progression. Other biomarkers for chonic kidney disease include: Urinary u-LFABp ( Liver-type fatty acid-binding protein); urinary NAG (N- Acetyl-b-O-glucosaminidase); urinary CTGF (Connective tissue growth factor); kidney tissue IL- 18 (Interleukin- 18);plasma ApoA-IV (Apo lipoprotein A-IV); urinary CD14 mononuclear cells; urinary and/or serum NGAL (Neutrophil gelatinase associated lipocalin); u-NCR (u- NGAL to creatinine ratio); eGFR: Estimated glomerular filtration rate; FGF-23: Fibroblast growth factor 23; CKD: Chronic kidney disease; serum KIM- 1 (Kidney injury molecule); AER (Albumin excretion rate;) GFR (Glomerular filtration rate); U-CGTF (Urinary- connective tissue growth factor). In some embodiments determination of urinary

trimethylamine and trimethylamine N-oxide is performed.

[0388] In some embodiments the efficacy of the genetically engineered bacteria or the extent of the disease is assessed using one or more biomarkers for atherosclerosis as described herein or known in the art (See e.g. Brown and Bittner Biomarkers of

Atherosclerosis: Clinical Applications Curr Cardiol Rep. 2008 Nov; 10(6): 497-504). Such biomarkers include but are not limited to, lipoproteins (such as apo lipoprotein (apo) B, low density lipoprotein (LDL), non-high-density lipoprotein cholesterol (HDL-C)), inflammatory biomarkers (such as Myeloperoxidase (MPO), Phospholipase A2 (Lp-PLA2), and C-Reactive protein (CRP)), coagulation markers (such as Plasma fibrinogen), cardiac troponin-I, Brain Natriuretic Peptide (BNP), and Cystatin C (a marker of renal function). In some embodiments determination of urinary trimethylamine and trimethylamine N-oxide is performed.

[0389] Trimethylaminuria is a rare inherited disorder due to decreased metabolism of dietary-derived trimethylamine by flavin-containing monooxygenase 3. Several single nucleotide polymorphisms of the flavin-containing monooxygenase 3 gene have been described and result in an enzyme with decreased or abolished functional activity for trimethylamine N-oxygenation thus leading to trimethylaminuria. In some embodiments, efficacy of the genetically engineered bacteria and or extent of disease in an animal model and/or a subject is evaluated using one or more biomarkers for Fish odor syndrome

(trimethylaminuria). In some embodiments determination of urinary trimethylamine and trimethylamine N-oxide is performed.

[0390] Trimethylamine-N-oxide (TMAO) levels can be determined by stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/MS/MS), for example, as described in Wang Z, et al., Measurement of trimethylamine-n-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry. Anal Biochem. 2014;455:35-40.

Methods of Screening

Generation of Bacterial Strains with Enhance Ability to Transport Metabolites or Biomolecules

[0391] Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses {e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

[0392] This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite or biomolecule.

[0393] For example, if the biosynthetic pathway for producing a certain metabolite or biomolecule is disrupted a strain capable of high-affinity capture of said metabolite or biomolecule can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid or metabolite, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the metabolite or biomolecule at regular intervals. Over time, cells that are most competitive for the metabolite or biomolecule - at growth-limiting concentrations - will come to dominate the population. These strains will likely have mutations in their metabolite- transporters resulting in increased ability to import the essential and limiting metabolite or biomolecule.

[0394] Similarly, by using an auxotroph that cannot use an upstream metabolite to form a certain metabolite or biomolecule, a strain can be evolved that not only can more efficiently imports the upstream metabolite, but also converts the metabolite into the essential downstream metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways.

[0395] A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound, this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate.

[0396] Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth- limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

[0397] Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

[0398] Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations "screened" throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 1011.2 CCD1. This rate can be accelerated by the addition of chemical mutagens to the cultures - such as N-methyl-N-nitro-N- nitrosoguanidine (NTG) - which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

[0399] At the conclusion of the ALE experiment, the cells are diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvo luted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. 0. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

[0400] Similar methods can be used to generate E. Coli Nissle mutants that consume or import trimethylamine (TMA).

Pharmaceutical Compositions and Formulations

[0401] Pharmaceutical compositions comprising the genetically engineered bacteria described herein may be used to treat, manage, ameliorate, and/or prevent disorders associated with trimethylamine and/or trimethylamine-N-oxide catabolism. Pharmaceutical compositions comprising one or more genetically engineered bacteria, alone or in

combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[0402] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic

modifications described herein, e.g., to express at least one trimethylamine catabolism gene or gene cassette. 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, e.g., to express at least one trimethylamine

catabolism gene(s) or gene cassette(s).

[0403] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into

compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of

administration.

[0404] The genetically engineered bacteria described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate-release, pulsatile-release, delay ed- release, or sustained release). Suitable dosage amounts for the genetically engineered

5 12 5

bacteria may range from about 10 to 10 bacteria, e.g., approximately 10 bacteria,

6 7 8 approximately 10 bacteria, approximately 10 bacteria, approximately 10 bacteria, approximately 10⁹ bacteria, approximately 10¹⁰ bacteria, approximately 10¹¹ bacteria, or approximately 10¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly.

[0405] The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal.

[0406] The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. The genetically engineered bacteria disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semisolid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the engineered bacteria may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.

[0407] The genetically engineered bacteria disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[0408] Tablets or capsules can be prepared by conventional means with

pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydro xypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., /actose, 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), hydro ymethylacry late- 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.

[0409] 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.

[0410] In some embodiments, enteric coating materials may be used, in one or more coating layers (e.g. , outer, inner and/o intermediate coating layers). Enteric coated polymers remain unionised at low pH, and therefore remain insoluble. But as the pH increases in the gastrointestinal tract, the acidic functional groups are capable of ionisation, and the polymer swells or becomes soluble in the intestinal fluid.

[0411] Materials used for enteric coatings include Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacry late), Cellulose acetate trimellitate (CAT), Poly( vinyl acetate phthalate) (PVAP) and Hydro xypropyl methylcellulose phthalate

(HPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers.

Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g. , maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylose acetate phthalate, cellulose acetate phthalate, hydro xyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate.

[0412] Coating polymers also may comprise one or more of, phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, Eudragit™ S (poly(methacrylic acid, methyl methacry late) 1 :2); Eudragit L100™ S (poly(methacrylic acid, methyl methacry late) 1 : 1); Eudragit L30D™, (poly(methacrylic acid, ethyl aery late) 1 : 1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate) l : l) (Eudragit™ L is an anionic polymer synthesized from methacry lie acid and methacrylic acid methyl ester), polymethyl methacrylate blended with acrylic acid and acrylic ester copolymers, alginic acid, ammonia alginate, sodium, potassium, magnesium or calcium alginate, vinyl acetate copolymers, polyvinyl acetate 30D (30% dispersion in water), a neutral methacrylic ester comprising poly(dimethylaminoethylacrylate) ("Eudragit E™), a copolymer of methylmethacrylate and ethylacrylate with trimethylammonioethyl

methacrylate chloride, a copolymer of methylmethacrylate and ethylacrylate, Zein, shellac, gums, or polysaccharides, or a combination thereof.

[0413] Coating layers may also include polymers which contain

Hydro xypropylmethylcellulose (HPMC), Hydro xypropylethylcellulose (HPEC),

Hydro xypropylcellulose (HPC), hydro xypropylethylcellulose (HPEC),

hydroxymethylpropylcellulose (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), hydro xymethylethylcellulose (HMEC), propylhydroxyethylcellulose (PHEC), methylhydroxyethylcellulose (M H EC),

hydrophobically modified hydroxyethylcellulose (NEXTON), carboxymethyl

hydroxyethylcellulose (CMHEC), Methylcellulose, Ethylcellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates such as ammonia alginate, sodium alginate, potassium alginate, acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydro xypropylcellulose phthalate (HPCP),

hydroxypropylethylcellulose phthalate (HPECP), hydroxyproplymethylcellulose phthalate (HPMCP), hydroxyproplymethylcellulose acetate succinate (HPMCAS).

[0414] 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); nonaqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The

preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria described herein.

[0415] In one embodiment, the genetically engineered bacteria of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et ah, Pediatrics, 134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to- swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

[0416] In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

[0417] In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, "flavor" is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

[0418] In certain embodiments, the genetically engineered bacteria may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its

inactivation.

[0419] In another embodiment, the pharmaceutical composition comprising the engineered bacteria may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria- fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the engineered bacteria are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the engineered bacteria are well known in the art. For example, see U.S. 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical

composition is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.

[0420] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[0421] The genetically engineered bacteria described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane,

trichlorofluoro methane, 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.

[0422] The genetically engineered bacteria may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0423] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

[0424] Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[0425] In other embodiments, 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. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly( methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly( vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[0426] Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by 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. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD₅₀, ED₅₀, EC₅₀, and IC₅₀ may be determined, and the dose ratio between toxic and therapeutic effects (LD₅₀/ED₅₀) may be calculated as 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. 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.

[0427] The pharmaceutical compositions 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 is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

Methods of Treatment

[0428] Another aspect of the disclosure provides methods of treating a disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental in a subject, or symptom(s) associated with the disorder in which trimethylamine and/or trimethylamine-N- oxide is detrimental in a subject. In one embodiment, the disorder in which trimethylamine is detrimental is a disorder associated with increased levels of trimethylamine. In one embodiment, a subject having a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental exhibits a daily urinary excretion of a ratio of less than 85% of TMAO/(TMAO+TMA) (see, for example, Wevers et al, 2008, Laboratory Guide to the Methods in Biochemical Genetics, Springer- Ver lag; New York, 2008, p. 781-792; and Mackay et ah, 2011, Clin. Biochem. Rev., 32(l):33-43), although other methods for diagnosing diseases or disorders associated in which trimethylamine and/or trimethylamine- N-oxide is detrimental are known in the art (see, for example, U.S. 2012/0157397, the entire contents of which are expressly incorporated herein by reference).

[0429] In one embodiment, a disease or disorder in which trimethylamine is detrimental is trimethylaminuria. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is a cardiovascular disease. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N- oxide is detrimental is kidney disease, such as chronic kidney disease. In another

embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is diabetes mellitus. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is insulin resistance. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is metabolic syndrome. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is nonalcoholic fatty liver disease. In another embodiment, a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental is nonalcoholic steatohepatitis.

[0430] The present disclosure surprisingly demonstrates that pharmaceutical compositions comprising the engineered bacterial cells of the invention may be used to treat disorders in which trimethylamine and/or trimethylamine-N-oxide is detrimental, such as cardiovascular disease, chronic kidney disease, and trimethylaminuria.

[0431] In one embodiment, the subject having a disease or disorder in which trimethylamine is detrimental has a mutation in a flavin-containing monooxygenase gene. In one embodiment, the flavin-containing monooxygenase gene is a FM03 gene.

[0432] In another aspect, the disclosure provides methods for decreasing the plasma level of trimethylamine and/or trimethylamine-N-oxide in a subject by administering a pharmaceutical composition comprising a bacterial cell of the invention to the subject, thereby decreasing the plasma level of trimethylamine and/or trimethylamine-N-oxide in the subject. In one embodiment, the subject has a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental. In another aspect, the invention provides methods for decreasing the level of trimethylamine and/or trimethylamine-N-oxide in the urine of a subject by administering a pharmaceutical composition comprising a bacterial cell of the invention to the subject, thereby decreasing the level of trimethylamine and/or trimethylamine-N-oxide in the urine of the subject. In one embodiment, the subject has a disease or disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental.

[0433] In some embodiments, the disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to chest pain, heart failure, or "fishy" smell. In some embodiments, the disease is secondary to other conditions, e.g., cardiovascular disease or kidney disease.

[0434] In certain embodiments, the bacterial cells of the invention are capable of catabolizing trimethylamine and/or trimethylamine-N-oxide in a subject in order to treat a disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental. In these embodiments, a patient suffering from a disorder in which trimethylamine and/or

trimethylamine-N-oxide is detrimental may be able to resume a substantially normal diet, or a diet that is less restrictive than a TMA-restricted or low TMA diet (for example, a diet low in TMA-containing nutrients, such as choline, phosphatidylcholine, and carnitine). In some embodiments, the bacterial cells may be capable of catabolizing trimethylamine and/or trimethylamine-N-oxide from additional sources, e.g., the circulating plasma or blood, in order to treat a disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental.

[0435] 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 disclosed herein are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria disclosed herein are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically. [0436] In certain embodiments, the pharmaceutical composition described herein is administered to reduce trimethylamine and/or trimethylamine-N-oxide levels in a subject. In some embodiments, the methods of the present disclosure reduce the trimethylamine and/or trimethylamine-N-oxide levels in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In another embodiment, the methods of the present disclosure reduce the trimethylamine and/or trimethylamine-N-oxide levels in a subject by at least two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten- fold. In another embodiment, the methods of the present invention increase the daily urinary excretion to a ratio of more than 85% of TMAO/(TMAO+TMA), more than 90% of TMAO/(TMAO+TMA), more than 92% of TMAO/(TMAO+TMA), or more than 95% of TMAO/(TMAO+TMA). In some embodiments, reduction is measured by comparing the trimethylamine and/or trimethylamine-N-oxide level in a subject before and after administration of the pharmaceutical composition. In one embodiment, the trimethylamine and/or trimethylamine-N-oxide level is reduced in the gut of the subject. In one embodiment, the trimethylamine and/or trimethylamine-N-oxide level is reduced in the urine of the subject. In another embodiment, the trimethylamine and/or trimethylamine-N-oxide level is reduced in the blood of the subject. In another embodiment, the trimethylamine and/or

trimethylamine-N-oxide level is reduced in the plasma of the subject. In another

embodiment, the trimethylamine and/or trimethylamine-N-oxide level is reduced in the fecal matter of the subject. In another embodiment, the trimethylamine and/or trimethylamine-N- oxide level is reduced in the sweat of the subject.

[0437] In one embodiment, the pharmaceutical composition described herein is administered to reduce trimethylamine and/or trimethylamine-N-oxide levels in a subject to normal levels. In another embodiment, the pharmaceutical composition described herein is administered to reduce trimethylamine and/or trimethylamine-N-oxide levels in a subject to below a normal level. In another embodiment, the pharmaceutical composition described herein is administered to increase the daily urinary excretion to a ratio of more than 92% of TMAO/(TM AO+TM A) .

[0438] In some embodiments, the method of treating the disorder in which

trimethylamine and/or trimethylamine-N-oxide is detrimental allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of treating the disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental allows one or more symptoms of the condition or disorder to improve by at least about two-fold, three-fold, fourfold, five-fold, six- fold, seven- fold, eight-fold, nine-fold, or ten-fold.

[0439] Before, during, and after the administration of the pharmaceutical

composition, trimethylamine and/or trimethylamine-N-oxide levels in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, peritoneal fluid, cerebrospinal fluid, fecal matter, 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, kidney, liver, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the disclosure to reduce levels of TMA. In some embodiments, the methods may include administration of the compositions of the disclosure to reduce TMA to undetectable levels in a subject. In some embodiments, the methods may include administration of the

compositions of the disclosure to reduce TMA concentrations to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the subject's TMA levels prior to treatment.

[0440] In some embodiments, the engineered bacterial cells of the invention produce at least one trimethylamine catabolism enzyme under exogenous environmental conditions, such as the low-oxygen environment of the mammalian gut, to reduce levels of

trimethylamine and/or trimethylamine-N-oxide in the urine, blood or plasma by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold as compared to unmodified bacteria of the same subtype under the same conditions.

[0441] Certain unmodified bacteria will not have appreciable levels of trimethylamine processing. In embodiments using genetically modified forms of these bacteria, processing of trimethylamine will be appreciable under exogenous environmental conditions.

[0442] Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) levels, or levels of TMA-containing compounds such as crotonobetaine, gamma-butyrobetaine, or carnitine, may be measured by methods known in the art. For example, plasma and urine TMA and TMAO levels can be measured using the assays described in U.S. Application Publication No. 2012/0157397, the entire contents of which are expressly incorporated herein by reference. In some embodiments, trimethylamine catabolism enzyme expression is measured by methods known in the art. In another embodiment, trimethylamine catabolism enzyme activity is measured by methods known in the art to assess TMA activity (see trimethylamine catabolism enzyme sections, supra).

[0443] 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 ah, 2009) or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the engineered bacteria may not colonize the subject and may be re- administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice can be determined. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

[0444] In one embodiments, the bacterial cells of the invention are administered to a subject once daily. In another embodiment, the bacterial cells are administered to a subject twice daily. In another embodiment, the bacterial cells are administered to a subject three times daily. In another embodiment, the bacterial cells are administered to a subject in combination with a meal. In another embodiment, the bacterial cells are administered to a subject prior to a meal. In another embodiment, the bacterial cells are administered to a subject after a meal. In another embodiment, the bacterial cells are not administered in the form of a food or edible product or incorporated into a food or edible product. 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 disease. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

[0445] The methods of the disclosed herein may comprise administration of a composition of the invention alone or in combination with one or more additional therapies, e.g., high blood pressure medicines or high cholesterol medicines. An important

consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the bacteria of the invention, e.g., the agent(s) must not interfere with or kill the bacteria.

[0446] The methods may further comprise isolating a plasma sample from the subject prior to administration of a composition of the invention and determining the level of the trimethylamine and/or trimethylamine N-oxide in the sample. In some embodiments, the methods of the invention may further comprise isolating a plasma sample from the subject after to administration of a composition of the invention and determining the level of trimethylamine and/or trimethylamine N-oxide in the sample. [0447] The methods of the disclosure may further comprise isolating a urine sample from the subject prior to administration of a composition of the invention and determining the level of trimethylamine and/or trimethylamine N-oxide in the sample. In some embodiments, the methods of the disclosure may further comprise isolating a urine sample from the subject after to administration of a composition of the invention and determining the level of trimethylamine and/or trimethylamine N-oxide in the sample.

[0448] In one embodiment, the methods of the disclosure further comprise comparing the level of the trimethylamine and/or trimethylamine N-oxide in the plasma sample from the subject after administration of a composition of the disclosure to the subject to the plasma sample from the subject before administration of a composition of the invention to the subject. In one embodiment, a reduced level of trimethylamine and/or trimethylamine-N- oxide in the plasma sample, or an increased level of TMAO (in conjunction with a corresponding decrease in TMA) in the plasma sample, from the subject after administration of a composition of the invention indicates that the plasma levels of TMA are decreased, thereby treating the disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental in the subject. In one embodiment, the plasma level of trimethylamine and/or trimethylamine-N-oxide or the urine level of trimethylamine and/or trimethylamine-N-oxide is decreased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the sample after administration of the pharmaceutical composition as compared to the level in the sample before administration of the pharmaceutical composition. In another embodiment, the level of TMA is decreased at least two-fold, three-fold, four- fold, or five-fold in the sample after administration of the pharmaceutical composition as compared to the level in the sample before administration of the pharmaceutical composition.

[0449] In one embodiment, the methods further comprise comparing the level of the trimethylamine and/or trimethylamine-N-oxide in the sample from the subject after administration of a composition to a control level of trimethylamine and/or trimethylamine- N-oxide.

[0450] The methods may further comprise isolating a sample from the subject prior to administration of a composition and determining the level of the trimethylamine and/or trimethylamine-N-oxide in the sample. In some embodiments, the methods may further comprise isolating a sample from the subject after to administration of a composition and determining the level of the trimethylamine and/or trimethylamine-N-oxide in the sample.

[0451] In one embodiment, the methods of the disclosure further comprise comparing the level of the TMA and/or TMAO in the urine sample from the subject after administration of a composition of the invention to the subject to the urine sample from the subject before administration of a composition of the disclosure to the subject. In one embodiment, a reduced level of TMA in the urine sample from the subject after administration of a composition of the invention indicates that the urine levels of TMA are decreased, thereby treating the disorder in which trimethylamine and/or trimethylamine-N-oxide is detrimental in the subject. In one embodiment, the urine level of trimethylamine and/or trimethylamine- N-oxide is decreased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the sample after administration of the pharmaceutical composition as compared to the urine level in the sample before administration of the pharmaceutical composition. In another embodiment, the urine level of the trimethylamine and/or trimethylamine-N-oxide is decreased at least two-fold, three-fold, four-fold, or five-fold in the sample after

administration of the pharmaceutical composition as compared to the level in the sample before administration of the pharmaceutical composition.

[0452] In one embodiment, the methods of the disclosure further comprise comparing the level of the TMA and/or TMAO in the plasma sample from the subject after

administration of a composition of the disclosure to a control level of TMA and/or TMAO.

Examples

[0453] The present disclosure is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents, and published patent applications, as cited throughout this application are hereby expressly incorporated herein by reference. It should further be understood that the contents of all the figures and tables attached hereto are also expressly incorporated herein by reference.

Development of Engineered Bacterial Cells

Example 1. Construction of Plasmids Encoding Trimethylamine Catabolism Enzymes

[0454] The trimethylamine dehydrogenase (SEQ ID NO: l), dimethylamine dehydrogenase (SEQ ID NO:2), and γ-glutamylmethylamide synthetase (SEQ ID NO:3) genes from Hyphomicrobium are synthesized (Genewiz), fused to the Tet promoter, cloned into the high-copy plasmid pUC57-Kan by Gibson assembly, and transformed into E. coli DH5a as described herein to generate the plasmid pTet-TMA. Example 2. Generation of Engineered Bacteria Comprising at Least One Trimethylamine Catabolism Enzyme

[0455] The pTet-TMA plasmid described above are transformed into E. coli Nissle, DH5a, or PIR1. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli (Nissle, DH5a or PIR1) is diluted 1: 100 in 4 niL of LB and grown until it reaches an OD₆oo of 0.4-0.6. lmL of the culture is then centrifuged at 13,000 rpm for 1 min in a 1.5mL microcentrifuge tube and the supernatant is removed. The cells are then washed three times in pre-chilled 10% glycerol and resuspended in 40uL pre-chilled 10% glycerol. The electroporator is set to 1.8kV. luL of a pTet-TMA miniprep is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled 1mm cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 500uL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing 50ug/mL Kanamycin for pTet-TMA.

Functional assays using recombinant bacterial cells of the invention

Example 3. Functional Assay Demonstrating that the Recombinant Bacterial

Cells of the Invention Decrease Trimethylamine Concentration

[0456] For in vitro studies, all incubations will be performed at 37° C. Cultures of E. coli Nissle containing pTet-TMA are grown overnight in LB and then diluted 1: 100 in LB. The cells are grown with shaking (250 rpm) to early log phase with the appropriate antibiotics. Anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of trimethylamine dehydrogenase, dimethylamine

dehydrogenase, and γ-glutamylmethylamide synthetase; and bacteria are grown for another 3 hours. Culture broths are then inoculated at 20% in flasks containing fresh LB culture media containing excess trimethylamine (TMA) and grown for 16 hours with shaking (250 rpm). A "medium blank" for each culture condition broth is also prepared whereby the "medium blank" is not inoculated with bacteria but treated under the same conditions as the inoculated broths. Following the 16 hour incubation period, broth cultures are pasteurized at 90°C for 15 minutes, centrifuged at 5,000 rpm for 10 minutes, and supernatants filtered with a 0.45 micron filter. [0457] Trimethylamine levels and activity in the supernatants is determined. Briefly, trimethylamine activity is determined spectrophotometrically by measuring the oxidation of NADPH at 340 nm as described by Boulton et al, 191 '4, J. Biochem., 140:253-263.

Trimethylamine N-oxide dimethylase activity is determined as described previously (Kim et al., 2001, Arch. Microbiol., 176:271-277). Moreover, trimethylamine-, dimethylamine- and methylamine dehydrogenase activities are determined spectrophotometrically by measuring the reduction of 2,6-chloramphenol-indophenol at 600 nm as described by Colby and Zatman, 1973, J. Biochem., 132: 101-112. Trimethylamine (TMA) degradation is also calculated by determining the concentration of trimethylamine in the inoculated media supernatant as compared to the equivalent reference "medium blank."

Example 4. In Vivo Studies Demonstrating that the Engineered Bacterial Cells of the Invention Decrease Trimethylamine Concentration

[0458] For in vivo studies, an atherosclerosis mouse model is used (see, for example Gregory et al., 2015, J. Biol. Chem., 290(9):2647-5660). Briefly, levels of trimethylamine and trimethylamine N-oxide are measured in the plasma and the urine of the mice prior to administration of the recombinant bacteria of the invention on day 0. On day 1, cultures of E. coli Nissle containing pTet-TMA are administered to three wild-type mice and three knockout mice once daily for a week. In addition, three knock-out mice are administered PBS as a control once daily for a week. Treatment efficacy is determined, for example, by measuring urine levels of TMA and TMAO and/or plasma levels of TMA and TMAO. A decrease in urine and/or plasma levels of trimethylamine, or an increase in the levels of

TMAO/(TMAO+TMA), after treatment with the recombinant bacterial cells indicates that the recombinant bacterial cells of the invention are effective for treating disorders in which trimethylamine is detrimental.

[0459] Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with disorders in which trimethylamine is detrimental, for example, decreased levels of aortic root atherosclerotic plaque, normal kidney structure, and lack of "fishy" smell, further indicates the efficacy of the recombinant bacterial cells of the invention for treating disorders in which trimethylamine (TMA) is detrimental. Example 5. In vivo efficacy of genetically engineered bacteria in TMAO induced renal dysfunction.

[0460] To determine the efficacy of the genetically engineered bacteria comprising one or more gene sequence(s) encoding one or more TMA catabolism enzymes for the treatment and/or prevention of renal dysfunction, a TMAO and/or choline fed mouse model is used essentially as described in Wang et al., Circ. Res. 2015 Jan 30; 116(3): 448-455.

[0461] E. coli Nissle 1917 comprising gene sequences for trimethylamine

dehydrogenase (SEQ ID NO: l), dimethylamine dehydrogenase (SEQ ID NO:2), and γ- glutamylmethylamide synthetase (SEQ ID NO:3) genes from Hyphomicrobium are under the control of an FNR promoter (SEQ ID NO: 27) are generated using the methods provided herein (hereinafter referred to as FNR-TMA). Conventionally housed 8-week old male mice (C57BL/6J background) are fed ad libitum a chemically defined diet comparable to normal chow (0.08gm% choline), or the same diet supplemented with either choline (1.0% final) or TMAO (0.12%) for six weeks. To test whether the genetically engineered FNR-TMA bacteria can ameliorate and/or prevent renal dysfunction in this model, separate groups of mice on each of the diets are either gavaged with 100 ul streptomycin resistant Nissle or the genetically engineered FNR-TMA strain once or twice daily for one to three weeks with various doses {e.g., ranging from 1χ10^Λ6 to 10xl0^A10 cells).

[0462] After 6 weeks, plasma TMAO levels of both groups of mice are measured, animals are sacrificed and Mason's trichome staining is performed on kidney sections to evaluate tubulointerstitital fibrosis and collagen deposition. Western analysis on kidney tissue lysates is conducted to assess phosphorylation levels of Smad3, an important regulator of the pro-fibrotic TGF-p/Smad3 signaling pathway during fibrotic kidney disease. Levies of kidney injury marker- 1 (KIM-1) are also evaluated by Western blotting.

[0463] In another study, the duration is extended to 16 weeks on normal and choline or TMAO chow and with or without dosing with streptomycin resistant Nissle and the engineered FNR-TMA strains, and serum cystatin C levels are measured and compared.

Example 6. In vivo efficacy of genetically engineered bacteria in TMAO induced atherosclerosis.

[0464] To determine the efficacy of the genetically engineered bacteria comprising one or more gene sequence(s) encoding one or more TMA catabolism enzymes for the treatment and/or prevention of renal dysfunction, a L-carnitine fed mouse model

(C57BL6J/ApoE-/- mice) is used essentially as described in Koeth et al. Nat Med. 2013 May; 19(5): 576-585. In this model, Apo lipoprotein E knockout mice on C57BL/6J background (C57BL/6J, Apoe-'-) mice are fed normal chow diet or the same diet is supplemented with L- carnitine from time of weaning.

[0465] C57BL/6J, Apoe^~'^~ are weaned at 28 days of age and placed on a standard chow control diet. L-carnitine is introduced into the diet by supplementing mouse drinking water with 1.3% L-carnitine , 1.3% L-carnitine for 15 weeks. To test whether the genetically engineered bacteria can ameliorate and/or prevent changes to cholesterol catabolism and atherosclerosis in this model, separate groups of mice on each of the diets are either gavaged with 100 ul streptomycin resistant Nissle or E. coli Nissle 1917 comprising gene sequences for trimethylamine dehydrogenase (SEQ ID NO: l), dimethylamine dehydrogenase (SEQ ID NO:2), and γ-glutamylmethylamide synthetase (SEQ ID NO:3) genes from Hyphomicrobium and gene sequence for trimethylamine permease from Methanosarcina barkeri (SEQ ID NO: 60) under the control of an FNR promoter (SEQ ID NO: 27) (hereinafter referred to as FNR- TMA/transporter) once or twice daily for one to fifteen weeks with various doses (e.g., ranging from 1χ10^Λ6 to 10xl0^A10 cells).

[0466] Mice are anaesthetized with Ketamine/Xylazine before terminal bleeding by cardiac puncture to collect blood. Mouse hearts are fixed and stored in 10% neutral buffered formalin before being frozen for sectioning. Aortic root slides are stained with Oil-red-0 and counterstained with Hematoxylin. The aortic root atherosclerotic lesion area is quantified as the mean of sequential sections of 6 microns approximately 100 microns apart.

[0467] Carnitine, TMA, and TMAO are determined using stable isotope dilution LC/MS/MS analysis of plasma recovered from mice at the time of sacrifice. Reverse cholesterol transport (RCT) is determined through 72 hour stool collection for

C57BL/6J, Apoe^~'^~ mice on normal chow or diet supplemented with either carnitine or choline, with or without oral gavage of streptopmycin resistant Nissle or the genetically engineered Nissle strain FNR-TM A/transporter.

Example 7. Generation of AThyA

[0468] An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine. [0469] A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a lOOum concentration are found in Table 11.

Table 11. Primer Sequences

[0470] For the first PCR round, 4x50ul PCR reactions containing lng pKD3 as template, 25ul 2xphusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions:

stepl: 98c for 30s

step2: 98c for 10s

step3: 55c for 15s

step4: 72c for 20s

repeat step 2-4 for 30 cycles

step5: 72c for 5min

[0471] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30ul nuclease free water.

[0472] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[0473] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by fit sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. {thyA auxotrophs will only grow in media supplemented with thy 3mM).

[0474] Next, the antibiotic resistance was removed with pCP20 transformation.

pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. lmL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in lmL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for lhours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and lOOug/ml carbenicillin and grown at 30°C for 16-24 hours. Next, transformants were colony purified no n- selectively (no antibiotics) at 42°C.

[0475] To test the colony-purified transformants, a colony was picked from the 42°C plate with a pipette tip and resuspended in ΙΟμί LB. of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.

[0476] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30ul nuclease free water.

[0477] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[0478] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. (thyA auxotrophs will only grow in media supplemented with thy 3mM).

[0479] Next, the antibiotic resistance was removed with pCP20 transformation.

pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. lmL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in lmL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for lhours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and lOOug/ml carbeniciUin and grown at 30°C for 16-24 hours. Next, transformants were colony purified no n- selectively (no antibiotics) at 42°C.

[0480] To test the colony-purified transformants, a colony was picked from the 42°C plate with a pipette tip and resuspended in

of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.

Example 8. Nitric oxide-inducible reporter constructs

[0481] ATC and nitric oxide-inducible reporter constructs were synthesized

(Genewiz, Cambridge, MA). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an

excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR- GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half- life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations (Fig. 24); promoter activity is expressed as relative florescence units.

[0482] An exemplary sequence of a nitric oxide-inducible reporter construct is shown, below. The nsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are Iboxedl. Table 12: SEQ ID NO: 46 ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgttgagcaggtcttgcagcgtgaaaccgt ccagatacgtgaaaaacgacttcattgcaccgccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttc gggcccatacactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgttcgggcggcgcggcca gcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccgcctttgaccagcgcggtaaccactttcatcaaatggctttt aaat cc ta tc a c at t c atatt acca c c tc tc tt ac c t ta at a ac c ca c

gacaattaatcatcggctcgtataatgtataacattcatattttgtgaattttaaactctagaaataattttgtttaactttaagaaggag atotocatotggctagcaaaggcgaagaattgttcacgggcgttgttcctattttgg

aaattcagcgttagcggcgaaggcgaaggcgatgctacgtatggcaaattgacgttgaaattcatttgtacgacgggcaaatt gcctgttccttggcctacgttggttacgacgttcagctatggcgttcaatgtttcagccgttatcctgatcatatgaaacgtcatga tttcttcaaaagcgctatgcctgaaggctatgttcaagaacgtacgattagcttcaaagatgatggcaattataaaacgcgtgct gaagttaaattcgaaggcgatacgttggttaatcgtattgaattgaaaggcattgatttcaaagaagatggcaatattttgggc cataaattggaatataattataatagccataatgtttatattacggctgataaacaaaaaaatggcattaaagctaatttcaaa attcgtcataatattgaagatggcagcgttcaattggctgatcattatcaacaaaatacgcctattggcgatggccctgttttgtt gcctgataatcattatttgagcacgcaaagcgctttgagcaaagatcctaatgaaaaacgtgatcatatggttttgttggaattc gttacggctgctggcattacgcatggcatggatgaattgtataaataataa

[0483] These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600= -0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units.

[0484] Fig. 24D shows a dot blot of NO-GFP constructs. E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1: 100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were

resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS -treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

[0485] Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR- inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1: 100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. Fig. 24 shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

Example 9. FNR promoter activity

[0486] In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9,

Cambridge, MA) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 3. The nucleotide sequences of these constructs are shown in Tables 12-16 (SEQ ID NO: 41-45). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity, exemplary results are shown in Fig. 23.

[0487] Table 13 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnrl (SEQ ID NO: 41). The construct comprises a translational fusion of the Nissle nirBl gene and the lacZ gene, in which the translational fusions are fused in frame to the 8th codon of the lacZ coding region. The Pfnrl sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0488] Table 14 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr2 (SEQ ID NO: 42). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8th codon of the lacZ coding region. The Pfnr2 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0489] Table 15 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr3 (SEQ ID NO: 43). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The Pfnr3 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0490] Table 16 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr4 (SEQ ID NO: 44). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The Pfnr4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0491] Table 17 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS (SEQ ID NO: 45). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrS l, fused to lacZ. The Pfnrs sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case. Table 13. Pfnrl-lacZ Construct Sequences

Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 41)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtc gtccggccttttcctctcttactctgctacgtacatctatttctataaatccgttcaatttg tctgttttttgcacaaacatgaaatatcagacaattccgtgacttaagaaaatttatacaaa tcagcaatataccccttaaggagtatataaaggtgaatttgatttacatcaataagcggggt tgctgaatcgttaaggtaggcggtaatagaaaagaaatcgaggcaaaaATGagcaaagtcag actcgcaattatGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCG TTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAG GCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTG GTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATA CTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTG ACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTC GCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATG GCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGC CGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGT GATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCG GCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTT ACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGG CGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCA GCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTC ACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCG TGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCG GTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATT CGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGAT GGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATC CGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCC AATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACC CGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCA TCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATC AAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCAC CGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGA AATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAA TATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCA GTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATG ATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGC CAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGC AAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCG AATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAG CCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACT GCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAAC CAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCG GAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGG AACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTC TTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACC CGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTG GGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGG CAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAA Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 41)

ACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGT GGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGG CGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGC CTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGT CTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGT GGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGC CATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGG GATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTC GCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 14. Pfnr2-lacZ Construct Sequences

Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 42)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatg catgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacaggagtat ttatattgcgcccgttacgtgggcttcgactgtaaatcagaaaggagaaaacacctATGacg acctacgatcgGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGT

TACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGG TTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATAC TGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGA CCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCG CTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGG CGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCC GTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTG ATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGG CATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTA CCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGC GAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAG CGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCA CACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGT GCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGG TTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTC GCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATG GTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCC GAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCA ATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCC GCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCAT CTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCA AATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACC GATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAA ATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAAT ATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAG TACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGA TGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCC AGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCA AAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGA Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 42)

ATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGC CGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTG CCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACC AAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGG AAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGA ACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCT TTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCC GTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGG GTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGC AGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAA CCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTG GATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGC GCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCC TTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTC TTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTG GCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCC ATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGG ATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCG CTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 15. Pfnr3-lacZ Construct Sequences

Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 43)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtc gtccggccttttcctctcttactctgctacgtacatctatttctataaatccgttcaatttg tctgttttttgcacaaacatgaaatatcagacaattccgtgacttaagaaaatttatacaaa tcagcaatataccccttaaggagtatataaaggtgaatttgatttacatcaataagcggggt tgctgaatcgttaaGGATCCctctagaaataattttgtttaactttaagaaggagatataca tATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGC CTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCG ATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAAC GTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTA CTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTG ATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGAC AGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGC GGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGA GCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAA GTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTA CGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCG CCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGC GTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTA TCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACG TCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTG ATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGAC GATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATT Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 43)

ATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAA GCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCT ACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGA TCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGG ATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGC CACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGC CGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGC GAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCG TCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAAT ATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGAT CGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGA AGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCA GCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGC AAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGA ACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGC AACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTG GCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAG CGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCT TTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTC ACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGC CTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCA CGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGG AAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAA TGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGC TGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGAC CGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTA CGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACC AGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACC AGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATAT GGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCG GTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 16. Pfnr4-lacZ construct Sequences

Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 44)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatg catgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacaggagtat ttatattgcgcccGGATCCctctagaaataattttgtttaactttaagaaggagatatacat

ATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGG CGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAG AGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCC TGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGA TACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACG TGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTAC TCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGA TGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACA Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 44)

GCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCG GTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAG CGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAG TTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTAC GGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGC CAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCG TCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTAT CGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGT CGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGA TTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACG ATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTA TCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAG CCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTA CCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGAT CATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGA TCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCC ACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCC GAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCG AATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGT CAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATA TGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATC GCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAA GCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAG CGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCA AGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAA CTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCA ACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGG CGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGC GGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTT TCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCA CCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCC TGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCAC GGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGA AAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAAT GTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCT GGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACC GCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTAC GTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCA GTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCA GCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATG GGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGG TCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA Table 17. Pfnrs-lacZ construct Sequences

Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 45)

GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgtaac aaaagcaatttttccggctgtctgtatacaaaaacgccgtaaagtttgagcgaagtcaataa actctctacccattcagggcaatatctctcttGGATCCctctagaaataattttgtttaact ttaagaaggagatatacatATGCTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCG

TGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCA GCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT GGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTG CGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATG CGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAG AATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCA GACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGG TCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCC GGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCA GGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGC AAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAG GCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCA GGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTG GCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCC GAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGA AGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGA ACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAG GTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGC CGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCC TGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACC GATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCG TAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACG ACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGC GGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCA GCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGC GCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAA TACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGA TCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTG GCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCG CATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCG AACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGA TGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAA GGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCT AACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCT GGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATC CCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATT TAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCC CGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACC CGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGC GGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACG CGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCAC GGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGAT TGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGC Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 45)

AAGAAAACTATCCCGACCGCCT TACTGCAGCCTGT T T TGACCGCTGGGATCTGCCAT TGTCA GACATGTATACCCCGTACGTCT TCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAAT T GAAT TATGGCCCACACCAGTGGCGCGGCGACT TCCAGT TCAACATCAGCCGCTACAGCCAAC AACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAAT ATCGACGGT T TCCATATGGGGAT TGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGA AT TCCAGCTGAGCGCCGGTCGCTACCAT TACCAGT TGGTCTGGTGTCAAAAATAA

Example 10. Nissle residence

[0492] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non- limiting example using a streptomycin- resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.

[0493] C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation {i.e., day 0), streptomycin-resistant Nissle (SYN-UCD103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 18. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

Table 18. CFU administered via oral gavage

[0494] On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs. 1-6; Table 19). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

[0495] Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 19.

[0496] Fig. 26 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

Table 19. Nissle residence in vivo

Example 11. Intestinal Residence and Survival of Bacterial Strains in vivo

[0497] Localization and intestinal residence time of streptomycin resistant Nissle, Fig. 27 was determined. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.

[0498] Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 10¹⁰ CFU/mL. Mice

(C57BL6/J, 10- 12 weeks old) were gavaged with 100 μΐ_^ of bacteria (approximately 10⁹ CFU). Drinking water for the mice was changed to contain 0.1 mg/mL anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post- gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.

[0499] In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37°C overnight, and colonies were counted. The amount of bacteria and residence time in each compartment is shown in Fig. 27.

Table 20. Informal Sequence Listing

CGTGACGCGAACACCAGTCATCGCTGGATCGAATTTGATTCTCTGGTCCTGG T GAC C GGC C GC C AT AGT GAGT GC AC C C T C TGGAAC GAAC T T AAAGC AC GT GA GAGC GAAT GGGC C GAAAAT GAT AT T AAGGGAAT C TAT C T GAT T GGC GAT GC C GAGGCTCCTCGTCTCATCGCCGATGCAACGTTTACGGGTCATCGTGTGGCAC GC GAAAT C GAAGAAGC AAAC C C AC AGAT C GC GAT C C C C T AC AAGC GT GAAAC GATCGCGTGGGGCACTCCCCACATGCCGGGCGGCAATTTCAAAATCGAGTAT AAGGTG

Dimethylamine ATGGCAAGAGATCCTCGCTTCGACATCCTCTTCACGCCACTGAAGCTCGGCT dehydrogenase CTAAAACGATACGTAATCGCTTCTATCAGGTGCCTCACTGCAACGGCGCGGG (Hyphomicrobium AACGAACTCGCCCGGCATGAACATGGCCCATCGCGGCATCAAGGCCGAAGGC dentrificans) GGCTGGGGCGCCGTCAACACTGAGCAGTGCTCGATCCATCCGGAATGCGACG

ACACGCTGCGCATCACGGCCCGTATCTGGGACCAGGGCGACATGCGCAACCT GCGCGCCATGGTCGACCACGTACACAGCCACGGCTCGCTCGCAGGCTGCGAA CTTTTCTACGGCGGACCGCACGCGCCGGCCATCGAATCGCGCACGATTTCGC GCGGTCCGAGCCAGTACAACTCCGAATTCGCGACTGTTCCCGGCTGCCCCGG C T T C AC C T AC AAT CAT GAAGC C GAC AT C GAC GAAC T C GAGC GC C T GC AGC AG CAGTATGTGGACGCGGCACTGCGCGCCCGCGATACCGGCTTCGACCTCGTGA ACGTCTACGGCGCTCACGCCTATGGCCCGATGCAGTGGCTCAACCCTTACTA CAACCGGCGCACAGACAAGTACGGCGGCAGCTTCGATAATCGCGCTCGCTTC T GGAT C GAGAC GC T T GAGAAGATCCGCC GGGC CGTCAAC GAT GAC GT GGC CC TTGTCACGCGCTGCGCGACCGACACCCTTTACGGAACGAAGGGCGTCGAGCT GACCGAGGACGGCCTGCGCTTCATCGAGCTTGCTTCGCCGTATCTCGACCTC TGGGACGTCAACATCGGCGACATCGCCGAGTGGGGTGAGGACGCGGGTCCCT CGCGCTTC TAT C C GAT C GC GC AC GAAAAC GAC T GGAT C C GC C AT AT C AAGC A GGCGACCAACAAGCCGGTCGTCGGCGTCGGCCGCTACTATGATCCGGAAAAG ATGCTGCAGGTCATCAAGGCGGGCATCATCGACATCATCGGCGCGGCGCGTC CGTCGATTGCCGATCCGTGGCTGCCACGCAAGATCGACGAGGGTCGCGTCGA CGATATCCGCACCTGCATCGGCTGCAACGTCTGCATCTCGCGCTGGGAAATG GGCGGCGTGCCGTTCATCTGCACGCAGAACGCAACGGCCGGCGAAGAGTATC GCCGCGGCTGGCATCCGGAGAAGTTCGAGCCGAAGAAGTCAGATCACGACGT CCTGATTGTTGGCGCCGGTCCTGCCGGGTCGGAATGCGCCCGCGTGCTCATG GAGCGCGGCTACACCGTGCATCTCGTCGATACGCGTGAAAAGACCGGCGGTT ATGTCAACGATGTCGCCACGCTGCCGGGTCTCGGCGAGTGGAGCTTCCATCG C GAC TAT C GC C AGAC GC AGC T C GAAAAGC T C C T C AAGAAGAAC C C T GAGT GC C AGAT C GC GC T C AAGC AGAAGC C GAT GAC T GC C GAT GAT AT T C T GC AGT AT G GCGCGTCGCGCGTCGTTATTGCGACGGGCGCGAAGTGGAGCACCACGGGCGT CAATCACCGCACGCACGAGCCGATCCCCGGCGCCGATGCGAGCCTGCCGCAC GTTCTGACACCCGAACAGGTGTACGAGGGCAAGAAGGCCGTCGGCAAGCGCG T GAT GAT CAT C AAC T AC GAT GC GT AC T AC AC AGC GC C C AGC C T T GC GGAGAA GTTCGCTCGCGCCGGTCACGACGTAACGGTTGCGACGGTGTGCGGTCTCGGC GCCTACATGGAGTACACCCTCGAGGGTGCGAACATGCAGCGCCTCATCCACG AGCTCGGCATCAAGGTTCTGGGCGAGACGGGTTGCTCGCGCGTCGAACAGGG CCGCGTCGAACTCTTCAACATCTGGGGTGAAGGCTACAAGCGCTCTTACAAG GGT GC C GGGC AGC T GC C GC GC AAC GA GAAC AC C AGC C AC GAAT GGC AT GAGT GCGATACCGTGATCCTCGTCACGTCGCGACGCTCCGAGGATACGCTCTATCG T GAAC TCAAGGCGCGGAAGGGC GAGT GGGAAGCAAAC GGC AT C AC GAAT GTC TTCGTCATCGGCGATGCCGAGTCGCCCCGTATCATCGCGGACGCGACGTTCG AC GGGC AT C GC C T GGC AC GC GAGAT C GAAGAT GC C GAT C C GC AGC AC C AGAA ACCGTACAAGCGCGAGCAGCGCGCCTGGGGCACGGCGTACAACCCGGACGAG AATCCGGATCTGGTGTGGCGCGTCTAG

γ-glutamylmethylamine ATGTCGCCAAGCGAGGCGCAGCAGTTTTTGAAAGAAAACCAGGTCAAATATA synthetase TCCTGGCTCAATTCGTAGACATCCACGGCTCAGCGAAAACTAAAAGCGTTCC (Methy overs tilis CGCGGAGCACTATAAAACGGTTGTCACCGATGGGGCGGGATTTGCGGGCTTT u iversalis) GCAATTTGGGGCATGGGTATGACCCCCAATGTTGACGCGGACTACATGGCGG

TGGGGGATGCATCAACCTTATCACTGGTGCCGTGGCAACCGGGCTACGCGCG CATCGCCTGTGATGGGCATACCCACGGCAAACCTCACGAGTACGATACGCGT GTTGTGTTAAAAAAACAGCTGGAGCAGATTACTGCCCGCGGCTGGACGTTCT TTACTGGTATGGAACCGGAGTTCAGTTTATTGCGTAAAGTAGAAGGTAAACT GCTGCCAGCGGATCCTGGTGATACGCTGAGCAAACCTTGTTACGACTATAAG GGTCTCAGCCGCGCGCGTGTGTTCTTAGAACGTTTATCAGAGAGTCTGCGCT C AGT T GGT AT C GAT GT GT AT C AGAT T GAT C AC GAAGAC GC GAAC GGC C AAT T T GAAAT CAAT T AC AC C T T T AC AGAT GC C T T GAC CAGCTGCGAC CAT TAT AC G TTTTTCAAAATGGGCGCTGCGGAAATCGCAGCGGAATTAGGGCTGATTTGCA GTTTTATGCCGAAACCCTTTAGCAATCGTCCGGGCAACGGTCTGCATATGCA TATGTCCATTGGGGATGGTAAACGTAATCTTTTTGAGGACAAAAGCGATAAA CACGGCCTTGCACTGTCTAAACTGGCCTACCATTGGGCCGCTGGGCTGCTTA AACACGCCCCGGCCCTGGCAGCGCTCTGTTGTCCGACGGTTAACAGCTATAA ACGCCTGGTAGTCGGCCGTAGTTTGACGGGTGCAACGTGGGCGCCTGCGTAT ATCTGCTATGGTGGTAACAATCGTAGCGGCATGATTCGCAGCCCGGGCGGGC GTCTCGAGTTACGCCTGCCTGATGCGTCTTGCAACGCGTATCTGGCCACAGC GGCTGTCATTGCGGCGGGTATGGACGGAGTGATTAATGAACTGGATCCCGGG GC T C C GC AGAAT GAC AAC T T AT AT GAAT AC AGC C AGGC GC AGC T GGAT GC C G CGGGAATCAAAGTACTGCCACAGAACTTGCACGAGGCGCTGCTGGCTCTGGA AAAAGATGAAGTGATTCGTAGCGCCCTTGGCCCGGTTGTGGATGAGTTTCTG CGCCTGAAACATATGGAATGGGTTGAATATATGCGTCACGTATCTGACTGGG AAGTCAATTCCTACCTGGAATTTTTT

FNR responsive GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTAT promoter #1 CGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAA

AT C C GT T CAAT TTGTCTGTTTTT T GC AC AAAC AT GAAAT AT CAGACAAT T C C GT GAC T T AAGAAAAT T T AT AC AAAT C AGC AAT AT AC C C C T T AAGGAGT AT AT AAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTA GGCGGT AAT AGAAAA GAAAT CGAGGCAAAA

FNR responsive ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTC promoter #2 AT GC AT GC AT C AAAAAAGAT GT GAGC T T GAT C AAAAAC AAAAAAT AT T T C AC

TCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCGACTGTAAATCA GAAAGGAGAAAAC AC C T

FNR responsive GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTAT promoter #3 CGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAA

AT C C GT T CAAT TTGTCTGTTTTT T GC AC AAAC AT GAAAT AT CAGACAAT T C C GT GAC T T AAGAAAAT T T AT AC AAAT C AGC AAT AT AC C C C T T AAGGAGT AT AT AAAGGT GAAT T T GAT T T AC AT C AAT AAGC GGGGT T GC T GAAT C GT T AAGGAT CCCTC T AGAAAT AAT T T T GT T T AAC T T T AAGAAGGAGAT AT AC AT

FNR responsive CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCT promoter #4 C AT GC AT GC AT C AAAAAAGAT GT GAGC T T GAT C AAAAAC AAAAAAT AT T T C A

C T C GAC AGGAGT AT T TAT AT T GC GC C CGGATCCC T C T AGAAAT AAT T T T GT T T AAC T T T AAGAAGGAGAT AT AC AT

FNR responsive AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTG promoter #5 TAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAGTTTG

AGC GAAGT C AAT AAAC T C T C T AC C CAT T C AGGGC AAT AT C T C T C T TGGATCC C T C T AGAAAT AAT T T T GT T T AAC T T T AAGAAGGAGAT AT AC AT

N-Methylglutamate ATGTGCGGAATCGTAGGCTTACTGGTTAAAACCCCCGCTCTGAAAGAACGCC Synthase T T GGT GAAC T GAT GGTGC CAAT GCT GAT TGGAAT GAC CGAACGCGGTCCGGA (subunit A) CAGCGCAGGCTTAGCAGTGTTTGGGGATTCTCTGGCGGACAACGCACGTAAG

(M e ihylo ve r sat His CTGAGTCTGTATTCCGGGCTTACCGATGATGGCGCTGATTTTAATTGGCACG universalis) GC C T GAC T C AC GC AT T GAAGGAGC AC C T GGAT GT C GAC GC C CAT AT C GAT GT

GAAGCATAAT CAC GC T GT GT TGTCGTTTGC GGT GAGC CCGGAACT GGT GAAG CGTTGGCTGCGTGAAAACCATCCGAAGCTGCACATTCTGTCTACAGGCCGTA C GAT C GAT C T C T AT AAGGAT AT T GGGAC AC C T GC T C AGGT T GC AGAGC GT T A CGATTTCAAGTCAATGAAAGGCTCTCACCTGGTTGGGCATACGCGCATGGCA ACTGAATCGGCCGTGACCCCAGACCGTGCCCACCCGTTTACGGCCGGGGAGG ATTTTTGCT TAGTT C AT AAT GGAT CAT T GAGT AAC C C GAAT T C TAT T C GT C G T AAAC T GAC C C C AGC C GGC AT C CAT T T T GAAAC T GAT AAC GAT AC C GAAGC A GCGTGTCGTTTCCTTGAGTGGCGCCTGCGCGAAGGTGATGATTTGGAAGTCG CGTTGCAAAAGGGGTTTGACGAGCTGGATGGCTTTTTTACCTTTCTGATGGG CAC GC C GGAAAAAC T GGC GC T TAT C C GT GAT CCGTTCGCCTGC AAGC CCGCC GTGGTTGCCGAAACCGATGACTATGTGGCGATTGCATCCGAATTTCGTAGCC T GGC GCAT C T GC C T GAC GT GAAAC AT GC GAAC GTGTTC GAAC C GGC AC CCGA GGAAATGTACGTTTGGAACGCA

N-Methylglutamate ATGACCTTTGATTTGGCATCGTCGAGTTTGACCGAAATGAATACGTTTCTTC Synthase ACAAAGGGCTGGAAGAAGGTGATAAACGCCATATCTCGGTGCTGAACCCGGA (subunit B) CGGCGCCCACAACATTGCGGTTGGTCTTAATCATCCAGTCACTGTTGAAGTC (M e thy love rsat is CATGGACATGCAGGCTATTATGCAGGCGGCATGAATAAACATGCCCGCGTGG universalis) TAATTCATGGCTCAGCCGGCACTGGTGTCGCCGAGAACATGATGAGCGGGAG

TGTTCATGTAAAGGGTTTTGCGTCCAACGGCGCCGGCGCCACCGCCCATGGC GGTCTGCTGGTCATTGACGGGGATGCAGGTCTGCGTTGCGGTATTTCCCTGA AAGGTGCGGATATCGTAGTCGGCGGCTCGGTCGGCTCGTTTTCAGGCTTCAT GGCTCAGGCGGGTCGCATGGTGATTTGTGGCGATGCTGGAGATGCTTTGGGG GACAGTCTGTATGAAGCGGTCATCTACCTGCGCGGAAATGTCAAATCCTTAG GGGC GGAT GC AC AGT T C GAGGAC AT GAC C GAC GC C GAT T AC GC AGT AC T C GC T GAAC T T T T AAGC AAAGC GGGT AT GGAT CAT GAC C C GAAAAGC T T T AAAC GT ATCGCTTCTGCGCGTACCCTGTATCATTGGAATGCTGATGCTAATCAGGAAT AT

N-Methylglutamate ATGGACATCAAACCTGTGTCGTTCAAACGTGTGAGTCGTGAGGAAAGTGCCA Synthase GCTTCGATCGCAGCACAATCGGCTACATTCAAAACGCCGCAGCGCATGGGCT (subunit C) TTATGAAATTCGTGGCATGGGCGCCAAACGCAAACTGCCCCATTTCGACGAC

(M e thy love rsatil is TTATTGTTCCTGGCGGGCTCGTTAAGCCGTTATCCACTGGAGGGTTATCGTG universalis) AGAAAT GC GT T AC GAAAAC CAT T C T CGGGAC T C GT T T C GC AAAGAAAC C CAT

CGAACTGGATATTCCGATCACAATCGCCGGCATGTCCTTCGGAGCCCTGTCG GCCAATGTTAAAGAAAGTTTAGGACGTGCAGCTACCGCGATGGGTACCACTA C GAC C AC T GGC GAC GGAGGT AT GAC C C C AGAAGAAC GT AAC AGC T CAAAAAC ACTGGTTTATCAGTGTCTCCCGTCCCGTTATGGGTTTAACCCGGACGACGTT C GT C GTGC GGAT GC GAT TGAAGTTGT TAT TGGTCAGGGTGCGAAGCCTGGAG GCGGGGGGATGCTGTTAGGGCAGAAAGT GAAC CCGCGTGT GGC GAAAATGCG TACACTGCCTCAGGGTGTGGATCAACGCTCAGCGTGTCGTCATCCCGATTGG AC AGGT C C T GAC GAT C T GGC GAT C AAGAT C C AGGAAT T AC GT GAAC T GAC GG ATTGGGAGAAGCCAATCTATGTTAAAGTTGGTGCGACCCGTACGTTTAATGA CGTGAAATTAGCCGTCCATGCCGGAGCGGACGTTGTGGTAGTCGACGGTATG CAAGGCGGCACCGCAGCGACGCAGACCTGCTATATCGAACATATCGGCATTC CAACGCTTGCCGCCGTACGTCAAGCAGTCGACGCACTGGAGGATCTGAACAT GAAGGGTCAAGTTCAGCTGATCGTTTCCGGCGGCATCCGCAGCGGAGCTGAT GTCGCGAAAGCCTTAGCTATGGGTGCCGACGCAGTTGCCATCGGCCAGGGTA TTTTGTACGCGTTGGGTTGTAATAGTGAGACCTACATTCAAGACGGCAAACA CATCAGCGCGCTGGAAGGTTACGATGCTCTGGGGACGCAGCCCGGTTTTTGT CACCATTGTCATACGGGTAAATGTCCTGTCGGCGTTACCACGCAGGACTCAG TTCTCGAACAGCGTCTTCAGCCTGATGTGGGCGCCCGTCGCGTTAAAAATTA TCTGAAAACGTTAAATATGGAGTTGACGACCATTGCGCGTGCATGCGGTAAA C AAAAT GT C C AC C AC T T GGAAC GC GAAGAC T T GGT T GC AC T GAC T C T T GAGG CCGCGGCGATGGCTCGTATCCCTCTGGCTGGTACTAGCTGGATTCCGGGTCA TAATGGCTAT

N-Methylglutamate CTGTCCAAGTCGCATCCGGAACCTCGTATGTTTACCGCCCACGATAAGTTGA Dehydrogenase AACCCAGCTACGACGTTGTTATCATCGGGGGCGGCGGTCATGGGCTGGCGTC Subunit A AGCTTATTATCTGGCACGTGACCATGGTATCACAAATGTAGCCGTGTTAGAA

(M e thy love rsatilis AAAGGATATATTGGTGGTGGCAATACGGGGCGCAACACTACTATCATCCGCT universalis C GAAC TAT C T GAC C C C GGAAGGGGT GAAAT T T T AC GAT AAAAGC GT T C AGC T

CTATCAAGACCTGTCGACGGAGTTTGATCTGAACTTGTTCTATTCGACCCGT GGGCACTTCACTCTGGCGCACACCGATAGCGCCATGCGCACCATGCGTTGGC GCGCTGAAGTCAATAAACACTACGGCATCGCTAGTGAAGTCGTGGGGCCTAA AGAAGTTAAACGTCAAACGCCTCAGATCGACCTGTCCTGTGGGGGTCATGCG CCAATTCAGGGCGCACTGTATCATGCACCGGGCGCAGTTGCTCGTCACGACG CTGTCGCATGGGGGTATGGGCGTGGCGCCGATATGCGCGGCGCGGAAATTCA TCAACAAACTGCGGTTACAGGCATTGAAGTCAAAGGGGGCAAAGTAGTCGGG GT AC AC AC AAC GAAAGGAT T CAT C AGC AC GAAC AAAGT GAT C T GC GC C GT AG CCGGCTTCACCCCGCGCATCACTGACATGGTCGGGTTCAAAACACCGATTTT CGTTCACCCGCTGCAAGCCATGGTATCGGAACCGATGAAACCTTGGCTGGAT ACGATCTTAGTTAGCGGCAGCCTGCATATTTACGTCTCCCAGTCGGCTCGTG GTGAACTCGTTATGGGAGCCTCGCTGGATCCGTATGAGGTTCAGTCTACCCG CTCGACTCTGGATTTCCCGGAAGGTCTTGCAGCGCACCTGCTGGATATGTTT CCTTTTTTAAGTCACAGCAAAGTAGTTCGCCAATGGGCGGGCATGGCCGACA TGACCCCAGACTTTGCGCCGATTATGGGCATGACGCCGGTTGAGGGTTTCTA CCTCGATTCAGGCTGGGGAACCTGGGGATTCAAAGCAACTCCGGTCTGCGGC AAGAC GAT GGC T TGGAC C GC T GT T AAC GAT AAAC C GC AT GAAC T GAT C AC GG GTTTCAGTTTAGATCGCTTCCGCAACTATTCTTTGACGGGTGAAAAAGGCGC AGCGTCAGTTGGGCATCATCTGGAGCGTGAAGATCTGGTGGCGTTAACCCTG GAAGCCGCCGCCATGGCTCGCATCCCGCTCGCGGGCACCTCCTGGATTCCGG GGCACAATGGGTAT

N-Methylglutamate ATGAAACTGATGACCTGCCCGATTAACGGAACGCGCCCAATTTCAGAGTTCG Dehydrogenase CTTATTGGGGTGAGATCCGCCCAGCCCCGGATGCTGACACGTGCTCAGATGA Subunit B TCAGTGGGCAGAATATGTCTTTCACCGTAACGGCGCACCAGGTGTTAAAAAG

{Meihyloversatilis GAGTGGTGGTGTCACACCCCATCGAACACTTGGTTTATCGCCGAACGTGATA universalis) C GGAGAAAGAC AAAGT AC T GC GT AC T TAT C T GC AC GGC GAT GAT AAA

N-Methylglutamate ATGCCTGATGAATGGCTGGATCGCAGTCGTACGGTGCGCTTCCGTTTCGAGG Dehydrogenase GTCGCTCTTTCGAAGGCCTGGCCGGCGATAGTATCGCTTCTGCTTTATGGGC Subunit C CGCGGGCCAGCGTAGCCAGGGGCGCTCATTTAAATATCATCGCGTCCGCGGC

(Meihyloversatilis ATTCTCTCGGCGGCAAACCATGACGTGAATGTCATGATGCAGGATGGCCCAA universalis) AATTGAATACCCGTGGTGATGTTGTGGCAGTCCGTGAAGGCATGGATCTGAC

GGCGGTTAACACGTTCGGTGGTTTAGCAAATGATAAAGCCCGTCACCTGAAC AAATTATCGCGCTTCTTGCCCGTGGGCTTTTACTATAAGGCGTTTCATAACA AGCGCCTGTTTCCCTTATGGGAGAAAATGTTCCGTCGCATCACGGGTTTGGG CGTTGTGGATTTCTCGACACCTCATATCCGTACTCAAAAACGTTACGACTTC TGCGATGTTCTGGTGATTGGCGCAGGGCCGAGCGGTTTGGCGGCTGCCTTGA GCGCAGCGGAGGCGGGAGCCTCAGTCGTTGTTTGCGATGAAAATGCTCGTGC GGGCGGCTCTGGGTTATTCCAGCTTGGTGCAGACGGTGCTCGTCGTCAGCAC ACCGTATCGCTCCTTCAGCAAGTTAAAGCCCACCCTCGTATCCGCCTGCTGG AAGGCACGTATGCGGGAGCTTACTACGCGGACCGCTGGGTGCCGTTGATTGA TGAGAACCGTATGACCAAAATGCGTGCTAAAAGTGTGATTGTGGCTTCGGGC GCATACGAACAGCCTTCAGTCTTTCGCAACAACGATCTGCCGGGTATTATGA ACGGCAGTGCCATGCAACGTCTTATCTACCGTTACGCGGTAAAACCATGTAA TACGGCAGTCGTGCTGGCAGCGAACGCTGATGGTTATCGTGTCGCTTTGGAT TTACTGGCGGCAGGGGCAGCCGTCGCGGCCGTGTTGGATATGCGCGCGTCTG TACCGGCAAGTCCGTTAGCGGATGCGCTGCGCGCCAAGGGTGTGGAAATCCT GGCGGGGCATGCAGTTTACGAAGCACATGCAGATGGCAAGGGGGAACTGGCC GGTGTTACTGCTTGTCGTATTGATGGCTCGGGGCGTGTTGTGAGTGGCTCGC AGCGTCGCATCAGCTGTGATGGCGTTGCGATGAGCACCGGCTGGAGCCCTGC CGCAAACCTGCTGTATCAGGCCGGAACCCGTATGCGTTTTGATGACTTGCTG CAGCAATTTGTGCCCGAACAACTGCCGGAGGGCGTTTTTGCGTGCGGCAAAG TTAATGGCGTGTTCCGTTTAGAATCCCGTATCACCGACGGTCAGCGTGCCGG GCTCGCCGCGGCCGCGAACGCGGGGTTTGAAGTTAGCGGTTCAATTACAGTC CCTGCGGAGACGGAGAGTCCAAGCCATGCATGGCCGGTTGTGGCCCATCCTG ACGGTAAAAATTTTGTGGACTTTGATGAAGACCTGCAAGTCAAAGATTTTGA AAAC GC T GT C C AAGAAGGC T AT GAC AAC AT C GAAC T T C T GAAAC GC T T T T C C ACGGTTGGTATGGGTCCCAGCCAGGGCAAACATAGCAACATGACTGCCTTGC GCATTCTGGCCCGTCTGACCGGCAAGTCACCCCAGCAGGTGGGTACGACAAC GGCCCGTCCGTTCTTCCATCCAGTCCCGTTAAGCCATCTGGCCGGCCGCGGT TTTTCGCCGCAACGCCACTCGCCGCTGCATGGCCGCCACGCGGCGTTGGGCG CCGTATTCATGCAAGCGGGAGCATGGGAACGCCCGGAGTATTATGCGGTGCC GGGAAAAAGTCGCATCGATTGCATCCGTGAAGAAGCACGCCGCGTTCGTACC GCTGTGGCCCTGATCGACGTGGGTACTTTAGGCAAACTGGAAATTCGCGGCC CGCAAGCGGGCGAATTTCTTAATCGTGTATATACCGGCCGTTACGATAACAT GAAGGTGGGGGCAACCCGTTACGCAGTAATGTGTGACGAATCAGGAGTCTTG TCGGATGAGGGTGTTGTCGCCCGCGTCGCGGATGATGTGTTCTATTTTACCA CCACCTCTTCAGGCGCCGCAACGGTATATCGCGAACTGAGCCGTCTGAATAT TGAGTGGAAACTGGATTGCGGCCTGATTAACTTGACCGGCTCCTACTCAGCA ATGAACCTGGCCGGGCCAGCAAGTCGCAAAGTCCTTGCGCAGCTGACGGATA TGGATCTCTCGAGCGCCGCGTTTCCTTATCTGGCCGTGCGCAGTGGGACCGT TGCGGGCATTCCTGCTCGTATGATGCGCGTGGGCTTTGTTGGCGAATGGGGC TGCGAGATTCATGCACCAGCAGAATACGGGGCAACCCTGTGGGATGCCCTGA TGAAAGCCGGCGAAAGTTCGGGGATCGGCCCGTTCGGCGTTGAGGCCCAGCG TTTGTTGCGTCTCGAAAAAGGCCATCTGATCGTGTCACAAGACACCGATGGT TTAACAAACCCGTTCGAAGTGGGTATGGATTGGGCCGTCAAGCTGGACAAAC CGTTCTTTACCGGTAAACGTTCTCTGCAAATTGTGCGTAAAATGCCACTGAA GCGCAAGCTGGTAGGGTTCCGCCTGGGGGAAAATCATTCAGGAGAAGTGCCT AAAGAGTGCCATTTAATCATCCAGGACGGGGACATTGCAGGTCGCGTCACCT CTATCTCGTGGTCTCCGCATGTGGGTCGTTTCATTGGCCTGGCGTTCGTCCT GCCGAGTATGAGCGAGACCGGTACTGCCTTTCAAATCCGCCTGACCGATGGT TCAATGGTGAACGCTGAAGTTTGCGATAGCCCATTTTTTGATCCGAAAGACG AAC GC C AGAAAGAAAT C GAA

N-Methylglutamate ATGCAGACCGTCGCTAGCTTTGGTAGTGCCGATGCCGCACGTTTGCCGCTTG Dehydrogenase CTGGTATTGGAGATTTGTCCTTTCGCCGTCGTGCTGGCGTGAAGGGTCCAGG Subunit D AGCCGCGGCGTGGCTGAATGCGTTAGGCATTGCAACGCCCGAACGGATGAAC

(M e thy love rsatilis TCCTGGTTGCGTATGGATGCCGGTACGCTGGTGCTGCGTTTAGGGAACACTG universalis ) AGTATCTTGTGGAAGATATGCCTGGCGGCGGTCGTACGGCGCAGATGTCCGC

AACTGCGCCGACGCATGGTGTGTACCCAGTTCCGCGTTACGATGCGGCCCTG ATTGTCGCTGGGCGCAACGCTCTGGAACTGACCCGCCAGACCTGCGCTTTCG ATTTTACAACCCTGAGCCCAGCAGCGCAGGGACTGGCAATGACCTCTATGGT TGGTGTCGGTATTACCGCCGTCGCCATGGAGAGCGGTGGTGAAACCTATTAT CGTTTGTGGTGCGATGGTACGTATGGTGGTTACTTATGGGCGACGCTCGTGG AAGTGGCGTCTGACTTAGGCGGCGGCGCGGTCGGACTGGAGTCATTAGGGCG TCTGGCCCAACAA

Formaldehyde ATGAAAGCGGTGGTATATCGCGGCCCACGCCAGGTTGCCATTGAAGACGTCC Dehydrogenase CGGAT C C CAAGAT C GAACGTC C AAC AGAT GC C AT TGTGAAAAT C AC AAGT AC

(Burkholderia sp.) AAATATTTGCGGCAGCGACCTGCATATGTATGAGGGCCGCACCGACTTTGAA

CAAGGTCGCATCTTTGGGCATGAAAACTTAGGAGTCGTGCAAGAAGTGGGCC CGGCCGTTGAACGCATTAAACCGGGTGATTGGGTGTGCTTACCGTTCAATGT GTCCTGTGGTCATTGCGCTAACTGCGAACGTGGCCTGACCGCCTTCTGTCTG AGCGCCAACCAGCCGGGCATTGCCGGCGGTGCTTTTGGCTTCGCGGATATGG GCCCGTGGGCAGGAGGCCAAGCGGAATACCTGCGTGTGCCATGGGCCGATTT TATGTCGCTGAAACTGCCGCCAGATGCGCAGGAAAAGCAGACGGATTACGTC ATGTGTGCAGACATTTTTCCAACAGGGTGGCATGCCACTGAACTGGCCGGTA TGCGTCCAGGCGATGCGGTCGTAATCTATGGTTCAGGGCCGGTTGGTTTGAT GGCCGCACATTCTGCTATGATTAAAGGCGCGCGCTCTGTAATGGTGGTCGAT TGTCACCCGGACCGTCTTAAACTGGCCGAATCTATTGGCGCTATCGCAATTG ACTATAGTAAAGAAGACCCAGTGCAACGCGTCATGGACCTTACGCGTGGTAT GGGCGCCGATGTCGGGTGTGAATGCGTGGGCTACCAGTGCCATGACCCGGCC C C GC AC C GC C AT GAGAAC C C GAAC C T GAC CAT GAATAAT C T GGT GGC AT C GG TGAAATTCACCGGCGGAATTGGTGTTGTTGGTGTGTTCGTGCCGGAAGATCC GGGCGCACAAGACGAATTAGCCAAACAGGGAAAGATCGCATTTGATTGGGGG AAATGTTGGTTCAAAGGACAACACATTGCTACCGGTCAATGTAACGTTAAAG CATACAATCGTCAACTGCGCGACCTGATTGATGCGGGTCGCGCAAAACCTAG C T T CAT C GT GAGC C AT GAAC T GAAAC T GGC GGAT GC GC C AGAT GC GT AT C AG CATTTCGATGTTCGCGAACATGGGTGGACTAAAGTTGTGCTGCATCCGGGGG GA

Methanol ATGACAAATTTTTTCATCCCTCCAGCCAGCGTGATCGGTCGCGGCGCCGTGA Dehydrogenase AAGAAGTGGGAACCCGCCTGAAGCAGATTGGTGCGAAAAAGGCCCTGATCGT

(Bacillus meihanolicus) GACTGATGCTTTCCTCCATTCAACTGGCCTGTCGGAAGAAGTCGCAAAAAAC

ATCCGTGAGGCAGGCTTGGATGTCGCAATTTTTCCGAAGGCTCAGCCGGATC CAGCGGATACTCAGGTCCATGAAGGGGTTGATGTATTTAAACAGGAAAACTG TGACGCGCTGGTGTCGATTGGTGGCGGGTCTTCCCATGACACCGCCAAAGCA ATTGGATTGGTCGCTGCGAACGGAGGCCGTATTAACGATTATCAGGGTGTCA ATAGCGTCGAAAAACCGGTCGTCCCGGTAGTAGCAATTACTACGACCGCGGG CACTGGCAGTGAAACGACGAGCCTCGCGGTTATCACTGATTCAGCCCGCAAA GTGAAGATGCCGGTAATTGATGAAAAAATTACCCCGACCGTGGCCATTGTGG ATCCAGAGCTGATGGTGAAAAAACCTGCCGGTCTGACGATCGCCACCGGCAT GGACGCGCTGAGTCATGCCATCGAAGCGTATGTCGCGAAAGGCGCAACCCCA GT GAC GGAT GC C T T T GC AAT C C AAGC C AT GAAAC T GAT TAAT GAGT AC C T T C CGAAAGCGGTGGCCAACGGCGAAGACATTGAAGCGCGTGAAGCGATGGCGTA CGCCCAGTATATGGCGGGTGTTGCCTTCAACAACGGGGGACTGGGTCTCGTC CATAGCATTTCTCATCAGGTGGGTGGTGTGTACAAGCTGCAACATGGTATTT GTAACTCTGTCAATATGCCTCATGTCTGTGCGTTCAACTTGATCGCAAAAAC CGAACGTTTCGCGCACATTGCCGAACTGCTGGGTGAAAACGTATCGGGTCTG AGCACCGCAGCCGCTGCCGAGCGCGCAATCGTCGCGCTGGAGCGTTACAACA AGAACTTCGGTATCCCCAGTGGTTATGCGGAAATGGGCGTCAAAGAAGAGGA CATTGAGCTGTTGGCGAAAAATGCGTTCGAAGATGTTTGTACTCAAAGCAAT CCGCGCGTCGCGACTGTGCAGGACATCGCCCAAATTATCAAAAACGCGCTC

Formate Dehydrogenase ATGAAGAAAATTGCGTCCGTCTGTCCTTACTGCGGTGCCGGTTGTAAATTGA {Enterobacter cloacae) ATTTAGTTGTGGAAAACAATCGTATTCTGCGCGCGGAAGCCGCGGAAGGCGT

GACCAACCAGGGCACGCTCTGTCTGAAGGGTTTTTACGGCTGGGACTTTCTG AATGATACCCGCCTTCTGACTCCTCGCTTGACCCAGCCGATGATTCGCTACC ACAAAGGCGAGGCGTTTACGCCCGTTACTTGGGAGGAAGCAATCCGCTACAC CGCTCACAAACTGCGCAGCATCAAAGAGCAGTATGGTCCACGCAGCATTATG ACGACCGGGAGCTCCCGTGGGACGGGTAACGAGACGAACTATGTGATGCAGA AGTTTGCTCGCGCAGTACTCAACACCAACAATGTAGATTGTTGTGCGCGTGT CTGTCATGGCCCGAGTGTTGCGGGCTTGCAGGAAACCCTGGGGAATGGTGCC ATGTCTAACTCGATTAACGACATTGAAAATTCGAAATGTCTGCTGGTGTTCG GCTACAACTGTGCCGATAGCCATCCGATCGTCGCACGCCGTGTGCTGAAAGC GCGTGAAAATGGTGCAAAAATCATTGTTTGCGATCCCCGCCGCATCGAAACC GCCCGTATCGCCGATCAACATCTGCAACTTAAAAATGGCTCGAATATGGCCC TGGTGAACGCGTTTGGTTACGTGCTGCTGGAAGAAGAGCTGTATGACAAAAA TTATGTGGCACGCTTCACTGAAGGGCTGGATGCATATCGTGAGACTGTGAAA GATTATGCGCCGGAAAACGTCGAGCATCTGACCGGCATCTCGGCGCGCGATG TTCGCCAAGCGATGCGCACTTTTGCCGCAGCGCCGAGCGCCACCGTGATGTG GGGGATGGGGGTCACACAGTTTGGCCAAGCTGTTGACGTGGTTAAAGGTTTA TCTTCCCTGGCGCTTTTAACCGGCAATCTTGGCCGTCCTGCTGTTGGAGTTG GCCCTGTTCGTGGGCAGAACAATGTTCAAGGCGCATGTGATATGGGCGTTCT GCCGAATATGTTTCCGGGATATCAGGATGTCACCGATCCCGCCGTGCGCCAA AAATTTGCCACGGCCTGGGGGATCGATGTGGGCATTATGGACAAAGAGGTGG GTACTCGCATCACGGAAGTACCACATTTAGCACTGGAAGGCAAAGTGAAGGC C TAT TAT AT TAT GGGC GAAGATCCGCTGCAAACGGAAGCGGATCTGGGACTG GTCCGTCGCGGCTTTGAGGCGCTCGATTTTGTGGTGGTTCAGGATATTTTTA TGACTAAAACCGCGGAGGTAGCCGATGTGCTTCTGCCTGCAACAAGTTGGGG AGAGCATGGCGGGGTCTTCACCTGCGCGGACCGTGGTTTCCAGCGCTTTGGA AAGGCGATTGAAGCGTCCGGCAATGTTAAACGTGACTGGGAAATTATCTCGC TCCTGGCGACGGAAATGGGGTATCCGATGCACTACACCAGTAACCAGCAGAT CTGGGACGAGATGCGTGAGTTGTGTCCCTTGTTTTACGGAGTGACCTACGAG AAAATGGGCGAAATGGGCCACGTGCAGTGGCCATGCCCGACATTAGATCACC C GGGT AC C C C C T AT T T AT AT AAAGAT AAC C AGT T C GAT AC T C C GT C GGGT AA AGGCCAATTGTTCGCTGCTGCATGGCGCGCGCCCGCGGAGATCCCCGATGAA ACTTACCCGTTGGTTCTGTGCACTGTTCGTGAAGTTGGTCACTACTCGTGCC GTTCGATGACAGGCAACTGTGCCGCGCTGCAGAGCTTAGCTGATGAACCGGG ACGTGTGCAGATGAATCCTTTGGATGCCGATTCTCTTGGAATCAAGGATGGA CAGTTAGTCTGGGTACATAGCCGCCGCGGTAAAGTGATTACCCGCGCATCAA TTAGCGAGCGCATCAACGCCGGAGCGGTCTACATGACGTATCAGTGGTGGAT TGGCGCGTGTAACGAGCTGACCCAAGATAACTTGGATCCGGTCAGTAAAACT C C GGAAAC C AAGT AC T GT GC AGT GAAAT T AGAGGC CAT T GAGGAT C AGC GT T GGGC GGAGGAT T T C GC AGC T T C T AGT TAT C AGAC GAT GAAAAC C C GC C T GAT TACGGCAGTAAATGTT

FNR responsive AT C C C CAT C AC T C T T GAT GGAGAT CAAT T C C C C AAGC T GC T AGAGC GT T AC C promoter #6 TTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCGACAGGCTCCCA

CAGGAGAAAACCG

FNR responsive CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACCTTGCCCTTA promoter #7 AAC AT T AGC AAT GT C GAT T TAT C AGAGGGC C GACAGGCT C C C AC AGGAGAAA

ACCG

nirBl GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTAT

CGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAA AT C C GT T CAAT TTGTCTGTTTTT T GC AC AAAC AT GAAAT AT C AGAC AAT T C C GT GAC T T AAGAAAAT T T AT AC AAAT C AGC AAT AT AC C C C T T AAGGAGT AT AT AAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTA GGCGGTAATAGAAAAGAAATCGAGGCAAAA

22. nirB2 CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTACAGCAAAC

GGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCC GTCACCGTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTT CTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAATATCAGAC AATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCATTAAGGA GTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTT AAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAAatgt11gt11aact11 aagaaggagatatacat

23. nirB3 GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCACTAT

CGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCTATAA ACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAATATCAGACAATTCC GTGACTTAAGAAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATAT AAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTA GGCGGTAATAGAAAAGAAATCGAGGCAAAA

24. ydfZ ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTC

ATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCAC TCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCGACTGTAAATCA GAAAGGAGAAAACACCT

25. nirB+RBS GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTAT

CGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAA ATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCC GTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAGGAGTATAT AAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGAT CCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

26. ydJZ+RBS CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCT

CATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCA CTCGACAGGAGTATTTATATTGCGCCCGGATCCCTCTAGAAATAATTTTGTT TAACTTTAAGAAGGAGATATACAT

27. fnrSl AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTG

TAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAGTTTG AGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

28. fnrS2 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTG

TAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTG AGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCC AAAGTGAACTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

29. nirB+crp TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGTCAGCA

TAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCGTC CGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCT CATTTTGTCTATTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTT AAGAAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATATAAAGGTG AATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGaaatgt gatctagttcacatttGCGGTAATAGAAAAGAAATCGAGGCAAAAatgtttg t t taact t t aagaaggagatatacat

30. fnrS+crp AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTG

TAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTG AGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCaaatgtga tctagttcacattt t t tgt t taact t t aagaaggagatatacat

31. katG TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACAGAGCACAA

AATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGTTATCAGCCTTGTTTTCT CCCTCATTACTTGAAGGATATGAAGCTAAAACCCTTTTTTATAAAGCATTTG TCCGAATTCGGACATAATCAAAAAAGCTTAATTAAGATCAATTTGATCTACA TCTCTTTAACCAACAATATGTAAGATCTCAACTATCGCATCCGTGGATTAAT TCAATTATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACTGTAGAG GGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAAGGTACC

32. dps TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTATCAATATAT

CTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACGCTTGTTACCACTATT AGTGTGATAGGAACAGCCAGAATAGCGGAACACATAGCCGGTGCTATACTTA ATCTCGTTAATTACTGGGACATAACATCAAGAGGATATGAAATTCGAATTCA TTAAAGAGGAGAAAGGTACC

ahpC GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATCCATGTCG

TTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGGCAGGCACTGAAGATA CCAAAGGGTAGTTCAGATTACACGGTCACCTGGAAAGGGGGCCATTTTACTT TTTATCGCCGCTGGCGGTGCAAAGTTCACAAAGTTGTCTTACGAAGGTTGTA AGGTAAAACTTATCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAA

ATTGGTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATGCGAATT CATTAAAGAGGAGAAAGGTACC

oxyS CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGCGATAGGTA

GAATAGCAATGAACGATTATCCCTATCAAGCATTCTGACTGATAATTGCTCA

CACGAATTCATTAAAGAGGAGAAAGGTACC

SR36 primer tagaactgatgcaaaaagtgctcgacgaaggcacacagaTGTGTAGGCTGGA

GCTGCTTC

SR38 primer gtttcgtaattagatagccaccggcgctttaatgcccggaCATATGAATATC

CTCCTTAG

SR33 primer caacacgtttcctgaggaaccatgaaacagtatttagaactgatgcaaaaag

SR34 primer cgcacactggcgtcggctctggcaggatgtttcgtaattagatagc

SR43 primer atatcgtcgcagcccacagcaacacgtttcctgagg

SR44 primer aagaatttaacggagggcaaaaaaaaccgacgcacactggcgtcggc

Pfhrl-lacZ construct, GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcgg low-copy cactatcgtcgtccggccttttcctctcttactctgctacgtacatctattt ctataaatccgttcaatttgtctgttttttgcacaaacatgaaatatcagac aattccgtgacttaagaaaatttatacaaatcagcaatataccccttaagga gtatataaaggtgaatttgatttacatcaataagcggggttgctgaatcgtt aaggtaggcggtaatagaaaagaaatcgaggcaaaaATGagcaaagtcagac tcgcaattatGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAA CCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGC

TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCA

GCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCC

GGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCC

TCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCT

ATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTG

TTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACG

CGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGC

GCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAG

CGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGG

AGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTT

TCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCA

AGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAA

GTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGT

GGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAAT

TATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTT

GAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGG

TTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGT

CGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAG

CCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTC

AGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAA

CAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTAC

ACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAA

CCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACC

CGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCG

AGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACG

ACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTA

TGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTAC

GCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCA

AAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATA

TGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCG

TTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATC

AGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGG

TGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTC TTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGC

AGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATA

CCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGAT

GGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTA

AGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACT

CTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCC

GGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGA

CACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGA

TTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGC

TTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGC

GCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGC

GACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCAT

TACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACG

CGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATT

TATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAAT

GTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGA

CCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCC

GCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGAT

CTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTC

TGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGA

CTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGC

CATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTT

TCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGA

ATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAA

TAA

Pfnr2-lacZ construct, GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgactt low-copy atggctcatgcatgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaat atttcactcgacaggagtatttatattgcgcccgttacgtgggcttcgactg taaatcagaaaggagaaaacacctATGacgacctacgatcgGGATCCTCTGG

CCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAA

TCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCC

CGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCT

TTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGA

TCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGT

TACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGC

CGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATAT

TGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTT

AACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGG

ACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGA

AAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAA

GATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGC

ATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGA

TGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTG

CGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCG

CCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTA

TGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGC

GCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACG

GCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGAT

TGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTT

AACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGA

TGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTG

TTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGC

CTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGA

ATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAAC

GCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTG

GGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCA

AATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACAC

CACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAG

CCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTG

GAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAG

TCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTA CAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATG

AAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAA

CGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCAT

CCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTAT

CCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAA

CGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGT

GAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTG

AACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGT

GCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAG

CAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACG

CCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAA

TAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATT

GGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGC

CGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGC

CTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTG

TTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCC

ACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCG

GATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGC

GATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCT

CAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCG

CCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTAT

ACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAAT

TGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCG

CTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCG

GAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCG

ACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCG

CTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

43. Pfnr3-lacZ construct GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcgg cactatcgtcgtccggccttttcctctcttactctgctacgtacatctattt ctataaatccgttcaatttgtctgttttttgcacaaacatgaaatatcagac aattccgtgacttaagaaaatttatacaaatcagcaatataccccttaagga gtatataaaggtgaatttgatttacatcaataagcggggttgctgaatcgtt aaGGATCCctctagaaataattttgtttaactttaagaaggagatatacatA TGACTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGA AAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCC

AGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGC

GCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGT

GCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTC

CCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGA

CCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGG

TTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAG

ACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACG

GGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCT

GAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGC

TGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCA

TTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTT

CCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCA

GAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTT

TGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGA

AATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAAC

GTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAG

TGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGA

CGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGC

AAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATG

GTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCA

GAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGG

TACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTG

AAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCT

ACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCAC

CCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATC

ACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACA GTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATG

TACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCA

TCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGA

ATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAG

GCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGG

ATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGG

CGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTG

GTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAAC

AGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGA

ATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTG

GATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAG

GTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACA

ACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAA

GCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCG

TGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAAC

GGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCA

GGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGC

TGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGA

AGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGC

CATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCG

ACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTT

ATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATC

AATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCC

TGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGG

GCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGG

GATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACG

GTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGG

CGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACC

AGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACG

GTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGC

GGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAA

AAATAA

Pfnr4-lacZ construct, GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgactt low-copy atggctcatgcatgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaat atttcactcgacaggagtatttatattgcgcccGGATCCctctagaaataat tttgtttaactttaagaaggagatatacat ATGACTATGATTACGGATTCTC TGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT

TAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAG

GCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGC

GCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTG

CGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCAC

GGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATC

CGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAA

TATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGC

GTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCC

AGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGG

AGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTG

GAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGC

TGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAA

TGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAG

CTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGG

TCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGG

TTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGG

AGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCG

ACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCG

GATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGC

GTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGA

CGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCG

CTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTAC

GGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAA

TGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGT AACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCG

CTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGA

TCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGA

CACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGAC

CAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGC

CTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAA

CAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGT

TTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATG

ATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCC

GAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCG

CATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTT

TATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGA

TAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGC

GGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGC

CTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGT

AGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGG

CAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCC

ACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGG

TAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGG

ATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTG

CGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAA

CGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCG

TTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCG

CCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTA

CCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCA

AGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGG

TCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGA

CCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATG

TATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCG

AATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAG

CCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCAC

GCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTG

GCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGG

TCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

45. Pfnrs-lacZ construct GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaa tggttgtaacaaaagcaatttttccggctgtctgtatacaaaaacgccgtaa agtttgagcgaagtcaataaactctctacccattcagggcaatatctctctt GGATCCctctagaaataattttgtttaactttaagaaggagatatacatATG

CTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAA

CCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGC

TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCA

GCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCC

GGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCC

TCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCT

ATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTG

TTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACG

CGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGC

GCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAG

CGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGG

AGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTT

TCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCA

AGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAA

GTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGT

GGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAAT

TATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTT

GAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGG

TTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGT

CGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAG

CCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTC

AGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAA

CAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTAC ACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAA CCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACC CGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCG AGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACG ACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTA TGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTAC GCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCA AAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATA TGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCG TTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATC AGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGG TGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTC TTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGC AGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATA CCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGAT GGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTA AGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACT CTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCC GGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGA CACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGA TTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGC TTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGC GCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGC GACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCAT TACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACG CGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATT TATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAAT GTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGA CCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCC GCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGAT CTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTC TGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGA CTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGC CATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTT TCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGA ATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAA

TAA

A nitric oxide-inducible ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtc reporter construct: ggcttgttgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacg The nsrR sequence is acttcattgcaccgccgagtatgcccgtcagccggcaggacggcgtaatcag bolded. The g/p gcattcgttgttcgggcccatacactcgaccagctgcatcggttcgaggtgg sequence is underlined. cggacgaccgcgccgatattgatgcgttcgggcggcgcggccagcctcagcc The PnsrR (NO cgccgcctttcccgcgtacgctgtgcaagaacccgcctttgaccagcgcggt regulated promoter and aaccactttcatcaaatggcttttggaaatgccgtaggtcgaggcgatggtg gcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgcgca

gtataacattcatattttgtgaattttaaactctagaaataattttgtttaa ctttaagaaggagatatacatatggctagcaaaggcgaagaattgttcacgg gcgttgttcctattttggttgaattggatggcgatgttaatggccataaatt cagcgttagcggcgaaggcgaaggcgatgctacgtatggcaaattgacgttg aaattcatttgtacgacgggcaaattgcctgttccttggcctacgttggtta cgacgttcagctatggcgttcaatgtttcagccgttatcctgatcatatgaa acgtcatgatttcttcaaaagcgctatgcctgaaggctatgttcaagaacgt acgattagcttcaaagatgatggcaattataaaacgcgtgctgaagttaaat tcgaaggcgatacgttggttaatcgtattgaattgaaaggcattgatttcaa agaagatggcaatattttgggccataaattggaatataattataatagccat aatgtttatattacggctgataaacaaaaaaatggcattaaagctaatttca aaattcgtcataatattgaagatggcagcgttcaattggctgatcattatca acaaaatacgcctattggcgatggccctgttttgttgcctgataatcattat ttgagcacgcaaagcgctttgagcaaagatcctaatgaaaaacgtgatcata tggttttgttggaattcgttacggctgctggcattacgcatggcatggatga attgtataaataataa

Trimethylamine MARDPKHDILFEPIQIGPKTLRNRFYQVPHCIGAGSDKPGFQSAHRSVKAEG dehydrogenase GWAALNTEYCSINPESDDTHRLSARIWDEGDVRNLKAMTDEVHKYGALAGVE (Methylophilus LWYGGAHAPNMESRATPRGPSQYASEFETLSYCKEMDLSDIAQVQQFYVDAA methylotrophus ) KRSRDAGFDIVYVYGAHSYLPLQFLNPYYNKRTDKYGGSLENRARFWLETLE

KVKHAVGSDCAIATRFGVDTVYGPGQIEAEVDGQKFVEMADSLVDMWDITIG DIAEWGEDAGPSRFYQQGHTIPWVKLVKQVSKKPVLGVGRYTDPEKMIEIVT KGYADI IGCARPSIADPFLPQKVEQGRYDDIRVCIGCNVCISRWEIGGPPMI CTQNATAGEEYRRGWHPEKFRQTKNKDSVLIVGAGPSGSEAARVLMESGYTV HLTDTAEKIGGHLNQVAALPGLGEWSYHRDYRETQITKLLKKNKESQLALGQ KPMTADDVLQYGADKVI IATGARWNTDGTNCLTHDP IPGADASLPDQLTPEQ VMDGKKKIGKRWILNADTYFMAPSLAEKLATAGHEVTIVSGVHLANYMHFT LEYPNMMRRLHELHVEELGDHFCSRIEPGRMEIYNIWGDGSKRTYRGPGVSP RDANTSHRWIEFDSLVLVTGRHSECTLWNELKARESEWAENDIKGIYLIGDA EAPRLIADATFTGHRVAREIEEANPQIAIPYKRETIAWGTPHMPGGNFKIEY KV

Dimethylamine MARDPRFDILFTPLKLGSKTIRNRFYQVPHCNGAGTNSPGMNMAHRGIKAEG dehydrogenase GWGAVNTEQCSIHPECDDTLRITARIWDQGDMRNLRAMVDHVHSHGSLAGCE (Hyphomicrobium LFYGGPHAPAIESRTISRGPSQYNSEFATVPGCPGFTYNHEADIDELERLQQ dentrificans) QYVDAALRARDTGFDLVNVYGAHAYGPMQWLNPYYNRRTDKYGGSFDNRARF

WIETLEKIRRAVNDDVALVTRCATDTLYGTKGVELTEDGLRFIELASPYLDL WDVNIGDIAEWGEDAGPSRFYP IAHENDWIRHIKQATNKPWGVGRYYDPEK MLQVIKAGI IDI IGAARPSIADPWLPRKIDEGRVDDIRTCIGCNVCISRWEM GGVPFICTQNATAGEEYRRGWHPEKFEPKKSDHDVLIVGAGPAGSECARVLM ERGYTVHLVDTREKTGGYVNDVATLPGLGEWSFHRDYRQTQLEKLLKKNPEC QIALKQKPMTADDILQYGASRWIATGAKWSTTGVNHRTHEPIPGADASLPH VLTPEQVYEGKKAVGKRVMI INYDAYYTAPSLAEKFARAGHDVTVATVCGLG AYMEYTLEGANMQRLIHELGIKVLGETGCSRVEQGRVELFNIWGEGYKRSYK GAGQLPRNENTSHEWHECDTVILVTSRRSEDTLYRELKARKGEWEANGITNV FVIGDAESPRI IADATFDGHRLAREIEDADPQHQKPYKREQRAWGTAYNPDE NPDLVWRV

γ-glutamylmethylamine MSPSEAQQFLKENQVKYILAQFVDIHGSAKTKSVPAEHYKTWTDGAGFAGF synthetase AIWGMGMTPNVDADYMAVGDASTLSLVPWQPGYARIACDGHTHGKPHEYDTR

(Methyioversatilis WLKKQLEQITARGWTFFTGMEPEFSLLRKVEGKLLPADPGDTLSKPCYDYK u iversalis) GLSRARVFLERLSESLRSVGIDVYQIDHEDANGQFEINYTFTDALTSCDHYT

FFKMGAAEIAAELGLICSFMPKPFSNRPGNGLHMHMSIGDGKRNLFEDKSDK HGLALSKLAYHWAAGLLKHAPALAALCCPTVNSYKRLWGRSLTGATWAPAY ICYGGNNRSGMIRSPGGRLELRLPDASCNAYLATAAVIAAGMDGVINELDPG APQNDNLYEYSQAQLDAAGIKVLPQNLHEALLALEKDEVIRSALGPWDEFL RLKHMEWVEYMRHVSDWEVNSYLEFF

N-Methylglutamate MCGIVGLLVKTPALKERLGELMVPMLIGMTERGPDSAGLAVFGDSLADNARK Synthase LSLYSGLTDDGADFNWHGLTHALKEHLDVDAHIDVKHNHAVLSFAVSPELVK (subunit A) RWLRENHPKLHILSTGRTIDLYKDIGTPAQVAERYDFKSMKGSHLVGHTRMA

(Methyioversatilis TESAVTPDRAHPFTAGEDFCLVHNGSLSNPNSIRRKLTPAGIHFETDNDTEA universalis) ACRFLEWRLREGDDLEVALQKGFDELDGFFTFLMGTPEKLALIRDPFACKPA

WAETDDYVAIASEFRSLAHLPDVKHANVFEPAPEEMYVWNA

N-Methylglutamate MTFDLASSSLTEMNTFLHKGLEEGDKRHISVLNPDGAHNIAVGLNHPVTVEV Synthase HGHAGYYAGGMNKHARWIHGSAGTGVAENMMSGSVHVKGFASNGAGATAHG (subunit B) GLLVIDGDAGLRCGISLKGADIWGGSVGSFSGFMAQAGRMVICGDAGDALG

(Methyioversatilis DSLYEAVIYLRGNVKSLGADAQFEDMTDADYAVLAELLSKAGMDHDPKSFKR u iversalis) IASARTLYHWNADANQEY

N-Methylglutamate MDIKPVSFKRVSREESASFDRSTIGYIQNAAAHGLYEIRGMGAKRKLPHFDD Synthase LLFLAGSLSRYPLEGYREKCVTKTILGTRFAKKPIELDIPITIAGMSFGALS (subunit C) ANVKESLGRAATAMGTTTTTGDGGMTPEERNSSKTLVYQCLPSRYGFNPDDV

(M e thylo ve r sat His RRADAIEWIGQGAKPGGGGMLLGQKVNPRVAKMRTLPQGVDQRSACRHPDW universalis) TGPDDLAIKIQELRELTDWEKPIYVKVGATRTFNDVKLAVHAGADWWDGM

QGGTAATQTCYIEHIGIPTLAAVRQAVDALEDLNMKGQVQLIVSGGIRSGAD VAKALAMGADAVAIGQGILYALGCNSETYIQDGKHISALEGYDALGTQPGFC HHCHTGKCPVGVTTQDSVLEQRLQPDVGARRVKNYLKTLNMELTTIARACGK QNVHHLEREDLVALTLEAAAMARIPLAGTSWIPGHNGY

N-Methylglutamate LSKSHPEPRMFTAHDKLKPSYDWI IGGGGHGLASAYYLARDHGITNVAVLE Dehydrogenase KGYIGGGNTGRNTTI IRSNYLTPEGVKFYDKSVQLYQDLSTEFDLNLFYSTR Subunit A GHFTLAHTDSAMRTMRWRAEVNKHYGIASEWGPKEVKRQTPQIDLSCGGHA

(Methyloversatilis P IQGALYHAPGAVARHDAVAWGYGRGADMRGAEIHQQTAVTGIEVKGGKWG universalis VHTTKGFISTNKVICAVAGFTPRITDMVGFKTPIFVHPLQAMVSEPMKPWLD

TILVSGSLHIYVSQSARGELVMGASLDPYEVQSTRSTLDFPEGLAAHLLDMF PFLSHSKWRQWAGMADMTPDFAPIMGMTPVEGFYLDSGWGTWGFKATPVCG KTMAWTAVNDKPHELITGFSLDRFRNYSLTGEKGAASVGHHLEREDLVALTL EAAAMARIPLAGTSWIPGHNGY

N-Methylglutamate

Dehydrogenase

MKLMTCPINGTRPISEFAYWGEIRPAPDADTCSDDQWAEYVFHRNGAPGVKK

Subunit B

EWWCHTPSNTWFIAERDTEKDKVLRTYLHGDDK

(Methyloversatilis

universalis)

N-Methylglutamate MPDEWLDRSRTVRFRFEGRSFEGLAGDSIASALWAAGQRSQGRSFKYHRVRG Dehydrogenase ILSAANHDVNVMMQDGPKLNTRGDWAVREGMDLTAVNTFGGLANDKARHLN Subunit C KLSRFLPVGFYYKAFHNKRLFPLWEKMFRRITGLGWDFSTPHIRTQKRYDF

(M e thy love rsatil is CDVLVIGAGPSGLAAALSAAEAGASVWCDENARAGGSGLFQLGADGARRQH universalis) TVSLLQQVKAHPRIRLLEGTYAGAYYADRWVPLIDENRMTKMRAKSVIVASG

AYEQPSVFRNNDLPGIMNGSAMQRLIYRYAVKPCNTAWLAANADGYRVALD LLAAGAAVAAVLDMRASVPASPLADALRAKGVEILAGHAVYEAHADGKGELA GVTACRIDGSGRWSGSQRRISCDGVAMSTGWSPAANLLYQAGTRMRFDDLL QQFVPEQLPEGVFACGKVNGVFRLESRITDGQRAGLAAAANAGFEVSGSITV PAETESPSHAWPWAHPDGKNFVDFDEDLQVKDFENAVQEGYDNIELLKRFS TVGMGPSQGKHSNMTALRILARLTGKSPQQVGTTTARPFFHPVPLSHLAGRG FSPQRHSPLHGRHAALGAVFMQAGAWERPEYYAVPGKSRIDCIREEARRVRT AVALIDVGTLGKLEIRGPQAGEFLNRVYTGRYDNMKVGATRYAVMCDESGVL SDEGWARVADDVFYFTTTSSGAATVYRELSRLNIEWKLDCGLINLTGSYSA MNLAGPASRKVLAQLTDMDLSSAAFPYLAVRSGTVAGIPARMMRVGFVGEWG CEIHAPAEYGATLWDALMKAGESSGIGPFGVEAQRLLRLEKGHLIVSQDTDG LTNPFEVGMDWAVKLDKPFFTGKRSLQIVRKMPLKRKLVGFRLGENHSGEVP KECHLI IQDGDIAGRVTSISWSPHVGRFIGLAFVLPSMSETGTAFQIRLTDG SMVNAEVCDSPFFDPKDERQKEIE

N-Methylglutamate

MQTVASFGSADAARLPLAGIGDLSFRRRAGVKGPGAAAWLNALGIATPERMN

Dehydrogenase

SWLRMDAGTLVLRLGNTEYLVEDMPGGGRTAQMSATAPTHGVYPVPRYDAAL

Subunit D

IVAGRNALELTRQTCAFDFTTLSPAAQGLAMTSMVGVGITAVAMESGGETYY

(M e thy love rsatilis

RLWCDGTYGGYLWATLVEVASDLGGGAVGLESLGRLAQQ

universalis )

Formaldehyde MKAWYRGPRQVAIEDVPDPKIERPTDAIVKITSTNICGSDLHMYEGRTDFE Dehydrogenase QGRIFGHENLGWQEVGPAVERIKPGDWVCLPFNVSCGHCANCERGLTAFCL

(Burkholderia sp. ) SANQPGIAGGAFGFADMGPWAGGQAEYLRVPWADFMSLKLPPDAQEKQTDYV

MCADIFPTGWHATELAGMRPGDAWIYGSGPVGLMAAHSAMIKGARSVMWD CHPDRLKLAESIGAIAIDYSKEDPVQRVMDLTRGMGADVGCECVGYQCHDPA PHRHENPNLTMNNLVASVKFTGGIGWGVFVPEDPGAQDELAKQGKIAFDWG KCWFKGQHIATGQCNVKAYNRQLRDLIDAGRAKPSFIVSHELKLADAPDAYQ HFDVREHGWTKWLHPGG

Methanol MTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVTDAFLHSTGLSEEVAKN Dehydrogenase IREAGLDVAIFPKAQPDPADTQVHEGVDVFKQENCDALVSIGGGSSHDTAKA

(Bacillus methanoUcus) IGLVAANGGRINDYQGVNSVEKPWPWAITTTAGTGSETTSLAVITDSARK

VKMPVIDEKITPTVAIVDPELMVKKPAGLTIATGMDALSHAIEAYVAKGATP VTDAFAIQAMKLINEYLPKAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLV HSISHQVGGVYKLQHGICNSVNMPHVCAFNLIAKTERFAHIAELLGENVSGL STAAAAERAIVALERYNKNFGIPSGYAEMGVKEEDIELLAKNAFEDVCTQSN PRVATVQDIAQ11KNAL Formate Dehydrogenase MKKIASVCPYCGAGCKLNLWENNRILRAEAAEGVTNQGTLCLKGFYGWDFL {Enterobacter cloacae) NDTRLLTPRLTQPMIRYHKGEAFTPVTWEEAIRYTAHKLRSIKEQYGPRSIM

TTGSSRGTGNETNYVMQKFARAVLNTNNVDCCARVCHGPSVAGLQETLGNGA MSNSINDIENSKCLLVFGYNCADSHPIVARRVLKARENGAKI IVCDPRRIET ARIADQHLQLKNGSNMALVNAFGYVLLEEELYDKNYVARFTEGLDAYRETVK DYAPENVEHLTGISARDVRQAMRTFAAAPSATVMWGMGVTQFGQAVDWKGL SSLALLTGNLGRPAVGVGPVRGQNNVQGACDMGVLPNMFPGYQDVTDPAVRQ KFATAWGIDVGIMDKEVGTRITEVPHLALEGKVKAYYIMGEDPLQTEADLGL VRRGFEALDFVWQDIFMTKTAEVADVLLPATSWGEHGGVFTCADRGFQRFG KAIEASGNVKRDWEI ISLLATEMGYPMHYTSNQQIWDEMRELCPLFYGVTYE KMGEMGHVQWPCPTLDHPGTPYLYKDNQFDTPSGKGQLFAAAWRAPAEIPDE TYPLVLCTVREVGHYSCRSMTGNCAALQSLADEPGRVQMNPLDADSLGIKDG QLVWVHSRRGKVITRASISERINAGAVYMTYQWWIGACNELTQDNLDPVSKT PETKYCAVKLEAIEDQRWAEDFAASSYQTMKTRLITAVNV

Trimethylamine

permease

{Methanosarcina MDLKTLKKQESKRKISKGYMWAFFCAIFWGIWYLPGTWWVLNPFDEMSSAI barken) AKTSGDGISLWTAVLITAFNALAVMLALMVWNGVLGKYGELVRTLKEFHPC

SKWFFLASIFGGPMAILGSFIAMGFIGGSFAAVAGLLYPWGSILAYYWYGE KISKRAAVGIAVIVLGGISIYGGGLFTELSSGNVPWIGYLGGLMAAAGWGIE GAIAGKGLDIAEPDAGLTLRFLGENI IWWI I IVPILALVGFPMYSFALQAFD PLTILILVFAGITFGFCYVTWYKSFPLIGVGRGQGIGNLYGLCAI IFLFLFF GDVPDWTIVIGGALCIAGSFVMFTEDASELETLRGE

Innielhyiamine

Permease

(Methanosarcina mazei) MGIDLKALKEQENRRRVSKGYMWALFCAVFWGIWYLPGTVIWVLNPFDEMFG

AIAETNGDGVSLWTAALITAFNALTVMLALMLWNGVLQKFGELSRTLREFS PCSKWFFLASVFGGPIAILGSFMAMGFIGGAFAAVAALLYPVIGSILAYYWY GEKITKRAAAGI IVI I IGGITIFGGGLITELGTGNVPWIGYLGGLMAAVGWG IEGAIAGKGLDISEPDVGLTLRFLGENI IWWI I IVPILAIVGFPMYTYALQA FEPMALLVLIFAGITFGFCYVSWYKSFPLIGVGRGQGIGNLYGFFAVIFIFL FFGDVPQWTILLGGTLCI IGSFVMFTEEAAEIETLRG

Trimethylamine

Permease

(M e thanosa rcina maze i) MEGFSVDLKALKKQESKRKISKGYMWALFCAVFWGIWYLPGTWWVLNPFDE

MYGAIAATNGDGVSLWTAVLITAFNALTVMLALMLWNGVLGKYGELGRTLK EFQPCSKWFFLASIFGGPMAILGSFIAMGFIGGAFAAVAGLLYPWGSILAY YWYGEKISKRAAIGIAVIVLGGISIYGGGLLTELSSGNIQWIGYLGGLMAAA GWGIEGAIAGKGLDIAEPDAGLTLRFLGENI IWWI I IVPILAIVGFPMYSFA FQAFEPLTLLVLVFAGITFGFCYVTWYKSFPLIGVGRGQGIGNLYGFCAI IF IFLFFGEVPQWTILVGGALCIAGSFIMFTEDASALETLRGE

Trimethylamine

permease {Methanolobus

psychrophilus R15) MDLQSLKKQENQRRIRKGFMWAFFCAILWGLWYIPGTWWVLNPFDEMYAEI

AATNGDGMSL11TAVLITSFNALTVILALMVWNGVLGKFGEMKRTIKEFNPC SKWFFLASIFGGPIAILGSFIAMGFVGGAFAAVAALMYPWGSILASKWYGE KISKRAFAGIVFI I IGGITIFGGGLLTELGAGNWWIGYIGGLMAAVGWGIE GAIAGKGLDIAEPDVGLTLRFLGENI IWWVIAVPILAILGFPMFKYALMVFE PLTMLVLVFAGITFGFCYVTWYKSFPLIGVGRGQGIGNLYGLFAWFIALFF GDIPSWTILVGGALCLIGSSIMFSEDSGQLESLRGE Trimethylamine

permease (Candidatus

Methanomassiliicoccus MIDVDLKEDSSPESPVQQDLEDARRLEHKKRVRFGYMMALFCAIFWGLWYVP intestirialis Issoire-Mxl ! GEAIWLINPFDQMTAAISATDGDALATVIGAWITGLNAIFVI IALLVWNGA

LGKLREMGRTVRQMRHCTKWFFLASIFGGPLAVLGSFLAMGFVGAAFAAVAG LMYPWGSILAHFTGQKVSKRALLGILAIVIGGITVYAGGLIEDLANGGGSI WGYVGGIMAAIGWGTEGI IASKGLDVAEPDIGITLRFVGEGI IWWVIAIPIL ALLGYPVLTYWQILDPLTIGVLIMLGITFGCCYVTWYKSFTLIGVNRGQGV GNLYGICAVIFLFVFMGRVPDWTI ILGGILCIVGSLVMFTEGSIGLETLKTD EFISKEAAPAKKTSSNTKKTNANGTASKVKLRPIKFRILELYSDGEEHWNSE WKVIQSEYGMGSGYGRDLINFDI IELAAGGLLKELAVAQDDGTYKAGALLH KYKITEFGKNQPCLASI

Claims

1. A bacterium comprising gene sequence(s) encoding one or more trimethylamine (TMA) catabolism enzyme(s) operably linked to a directly or indirectly inducible promoter that is not associated with the TMA catabolism enzyme gene in nature.

2. The bacterium of claim 1, wherein the trimethylamine (TMA) catabolism enzyme catabolizes trimethylamine (TMA) and/or trimethylamine N-oxide.

3. The bacterium of claim 1 or claim 2, wherein the bacterium further comprises gene sequence(s) encoding one or more transporter(s) of trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) operably linked to a promoter that is not associated with the transporter gene in nature.

4. The bacterium of claim 3, wherein the promoter that is not associated with the transporter gene in nature is a directly or indirectly inducible promoter.

5. The bacterium of claim 3 or claim 4, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme and the promoter operably linked to the gene sequence(s) encoding the transporter of trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) are separate copies of the same promoter.

6. The bacterium of claim 3 or claim 4, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme and the promoter operably linked to the gene sequence(s) encoding the transporter of TMA and/or TMAO are the same copy of the same promoter.

7. The bacterium of claim 3 or claim 4, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme and the promoter operably linked to the gene sequence(s) encoding the transporter of TMA and/or TMAO are different promoters.

8. The bacterium of any one of claims 1-7, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.

9. The bacterium of any one of claims 1-7, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme is directly or indirectly induced by exogenous environmental conditions found in the small intestine.

10. The bacterium of any one of claims 1-9, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme is directly or indirectly induced under low-oxygen or anaerobic conditions.

11. The bacterium of any one of claims 1-10, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR- responsive promoter.

12. The bacterium of any one of claims 1-11, wherein the promoter operably linked to the gene sequence(s) encoding the trimethylamine catabolism enzyme is an FNRS promoter.

13. The bacterium of any one of claims 2-12, wherein the promoter operably linked to the gene sequence(s) encoding the transporter of TMA and/or TMAO is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.

14. The bacterium of any one of claims 2-13, wherein the promoter operably linked to the gene sequence(s) encoding the transporter of TMA and/or TMAO is directly or indirectly induced under low-oxygen or anaerobic conditions.

15. The bacterium of any one of claims 2-14, wherein the promoter operably linked to the gene sequence(s) encoding the transporter of TMA and/or TMAO is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR- responsive promoter.

16. The bacterium of any one of claims 1-15, wherein the gene sequence(s) encoding the trimethylamine catabolism enzyme is located on a chromosome in the bacterium.

17. The bacterium of any one of claims 1-16, wherein the gene sequence(s) encoding the trimethylamine catabolism enzyme is located on a plasmid in the bacterium.

18. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s) that convert trimethylamine to γ-glutamylmethylamide.

19. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding one or more trimethylamine catabolism enzyme(s) that convert trimethylamine into carbon dioxide and NADH.

20. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a trimethylamine dehydrogenase.

21. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a dimethylamine dehydrogenase.

22. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a γ-glutamylmethylamine synthetase.

23. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a N-methylglutamate synthase.

24. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a N-methylglutamate dehydrogenase.

25. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a formaldehyde dehydrogenase.

26. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a formate dehydrogenase.

27. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a methanol dehydrogenase.

28. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a methylamine-glutamate N-methyltransferase.

29. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, and a γ-glutamylmethylamine synthetase.

30. The bacterium of claim 29, wherein the bacterium further comprises gene sequence(s) encoding an N-methylglutamate synthase, an N-methylglutamate dehydrogenase, a formaldehyde dehydrogenase, and a formate dehydrogenase.

31. The bacterium of claim 30, wherein the bacterium further comprises a methanol dehydrogenase.

32. The bacterium of any one of claims 1-17, wherein the bacterium comprises gene sequence(s) encoding a trimethylamine dehydrogenase, a dimethylamine dehydrogenase, a methylamine-glutamate N-methyltransferase, and an N-methylglutamate dehydrogenase.

33. The bacterium of claim 32, wherein the bacterium further comprises gene sequence(s) encoding a formaldehyde dehydrogenase and a formate dehydrogenase.

34. The bacterium of claim 33, wherein the bacterium further comprises a methanol dehydrogenase.

35. The bacterium of any one of claims 1-34, wherein the bacterium is a probiotic bacterium.

36. The bacterium of any one of claims 1-34, wherein the bacterium is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus and Lactococcus.

37. The bacterium of claim 36, wherein the bacterium is of the genus Escherichia.

38. The bacterium of claim 37, wherein the bacterium is of the species Escherichia coli strain Nissle.

39. The bacterium of any one of claims 1-38, wherein the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.

40. The bacterium of claim 39, wherein the bacterium is an auxotroph in diaminopimelic acid or an enzyme in the thymine bio synthetic pathway.

41. The bacterium of any of claims 1-40, wherein the bacterium is further engineered to harbor a gene encoding a substance that is toxic to the bacterium, wherein the gene is under the control of a promoter that is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut.

42. A pharmaceutical composition comprising the bacterium in any of claims 1-41, and a pharmaceutically acceptable carrier.

43. The pharmaceutical composition of claim 42 formulated for oral administration.

44. A method for treating a disease or disorder in which trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) is detrimental in a subject, the method comprising administering a pharmaceutical composition of claim 42 or claim 43 to the subject, thereby treating the disease or disorder in which trimethylamine is detrimental in the subject.

45. A method for decreasing the level of trimethylamine (TMA) and/or trimethylamine N-oxide (TMAO) in the gut of a subject, the method comprising administering a

pharmaceutical composition of claim 42 or claim 43 to the subject, thereby decreasing the level of trimethylamine and/or TMAO in the gut of the subject.

46. The method of claim 44, wherein the disorder in which trimethylamine is detrimental is trimethylaminuria.

47. The method of claim 44, wherein the disorder in which trimethylamine is detrimental is a cardiovascular disease.

48. The method of claim 47, wherein the cardiovascular disease is atherosclerosis.

49. The method of claim 44, wherein the disorder in which trimethylamine is detrimental is kidney disease.