EP3328988A1 - Bacteria engineered to treat disorders involving propionate catabolism - Google Patents

Bacteria engineered to treat disorders involving propionate catabolism

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Publication number
EP3328988A1
EP3328988A1 EP16751733.3A EP16751733A EP3328988A1 EP 3328988 A1 EP3328988 A1 EP 3328988A1 EP 16751733 A EP16751733 A EP 16751733A EP 3328988 A1 EP3328988 A1 EP 3328988A1
Authority
EP
European Patent Office
Prior art keywords
gene
propionate
bacterium
encoding
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16751733.3A
Other languages
German (de)
English (en)
French (fr)
Inventor
Dean Falb
Paul Miller
Alex TUCKER
Jonathan Kotula
Vincent ISABELLA
Yves Millet
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Synlogic Operating Co Inc
Original Assignee
Synlogic Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2016/032565 external-priority patent/WO2016183532A1/en
Priority claimed from PCT/US2016/037098 external-priority patent/WO2016201380A1/en
Application filed by Synlogic Inc filed Critical Synlogic Inc
Publication of EP3328988A1 publication Critical patent/EP3328988A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • propionyl CoA carboxylase PCC
  • MUT methylmalonyl CoA mutase
  • Enzyme deficiencies or mutations which lead to the toxic accumulation of propionyl CoA or methylmalonyl CoA result in the development of disorders associated with propionate catabolism, such as Propionic Acidemia (PA) and Methylmalonyl Acidemia (MMA). Severe nutritional deficiencies of Vitamin B12 can also result in MMA
  • immunosuppressant therapy limit the practicality of liver transplantation for treatment of disorders involving the catabolism of propionate. Therefore, there is significant unmet need for effective, reliable, and/or long-term treatment for disorders involving the catabolism of propionate.
  • the present disclosure provides engineered bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating disorders involving the catabolism of propionate.
  • the engineered bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, one or more propionate catabolism genes to treat the disease, as well as other optional circuitry designed to ensure the safety and non-colonization of a subject that is administered the engineered bacteria, such as, for example, auxotrophies, kill switches, and combinations thereof.
  • engineered bacteria are safe and well tolerated and augment the innate activities of the subject’s microbiome to achieve a therapeutic effect.
  • the disclosure provides a bacterial cell that has been genetically engineered to comprise one or more genes, gene cassettes, and/or synthetic circuits encoding a propionate catabolism enzyme or propionate catabolism pathway, and is capable of metabolizing propionate and/or other metabolites, such as propionyl CoA, methylmalonate, and/or methylmalonyl CoA.
  • the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells may be used to treat and/or prevent diseases associated with propionate catabolism, such as propionic acidemia (PA) and methylmalonic acidemia (MMA).
  • PA propionic acidemia
  • MMA methylmalonic acidemia
  • the disclosure provides a bacterial cell that has been engineered to comprise gene sequence(s) encoding one or more propionate catabolism enzyme(s). In some embodiments, the disclosure provides a bacterial cell has been engineered to comprise gene sequence(s) encoding one or more propionate catabolism enzyme(s) and is capable of reducing the level of propionate and/or other metabolites, for example, methylmalonate, propionyl CoA and/or methylmalonyl CoA. In some embodiments, the disclosure provides a bacterial cell has been engineered to comprise gene sequence(s) encoding one or more propionate catabolism enzyme(s) that is operably linked to an inducible promoter.
  • the disclosure provides a bacterial cell has been engineered to comprise gene sequence(s) encoding one or more propionate catabolism enzyme(s) that is operably linked to an inducible promoter that is induced under low oxygen and/or anaerobic conditions, e.g., such as those conditions found in the mammalian gut.
  • the disclosure provides a bacterial cell has been engineered to comprise gene sequence(s) encoding one or more propionate catabolism enzyme(s) that is operably linked to an inducible promoter that is induced by environmental signals and/or conditions found in the mammalian gut (e.g., induced by metabolites or biomolecules found in the mammalian gut).
  • the disclosure provides a bacterial cell has been engineered to comprise gene sequence(s) encoding one or more propionate catabolism enzyme(s) and is capable of reducing the level of propionate and/or other metabolites, for example, methylmalonate, propionyl CoA and/or methylmalonyl CoA in low-oxygen environments, e.g., the gut.
  • the bacterial cell has been genetically engineered to comprise one or more circuits encoding one or more propionate catabolism enzyme(s) and is capable of processing and reducing levels of propionate, methylmalonate, propionyl CoA and/or methylmalonyl CoA, e.g., in low-oxygen environments, e.g., the gut.
  • the bacterial cell of the disclosure has also been genetically engineered to comprise gene sequence(s) encoding one or more transporter(s) of propionate.
  • the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells of the disclosure may be used to convert excess propionic acid, propionyl CoA, and/or methylmalonyl CoA into non-toxic molecules in order to treat and/or prevent conditions associated with disorders involving the catabolism of propionate, such as
  • FIG.1 depicts schematics of exemplary synthetic biotics of the disclosure for the treatment of propionic acidemia and/or methylmalonic acidemia and/or disorders characterized by propionic acidemia and/or methylmalonic acidemia.
  • FIG.1A depicts a schematic of an exemplary synthetic biotic of the disclosure comprising a gene cassette expressing the prpE, phaB, phaC, and phaA genes under the control of an inducible promoter. PrpE, PhaB, PhaC, and PhaA are capable of catabolizing propionate or propionyl CoA and/or methylmalonic acid or methylmalonyl CoA into P(HV-co-HB).
  • FIG.1B depicts a schematic of an exemplary synthetic biotic of the disclosure comprising a gene cassette expressing prpE, accA, pccB, mmcE, mutA and mutB as two polycistronic messages from two inducible promoters.
  • PrpE, accA, pccB, mmcE, mutA and mutB are capable of catabolizing propionate or propionyl CoA and/or methylmalonic acid or methylmalonyl CoA into succinate, which can be utilized through the TCA cycle or exported from the cell.
  • Protein lysine acyltransferase (pka) is deleted to prevent inactivation of PrpE.
  • FIG.2 depicts various branched chain amino acid (BCAA) degradative pathways and the metabolites and associated diseases relating to BCAA metabolism.
  • BCAA branched chain amino acid
  • FIG.3 depicts the cause and symptoms of a disease associated with propionate catabolism, such as Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA), which result from genetic defects in propionyl-CoA carboxylase or methylmalonyl- CoA mutase.
  • PA Propionic Acidemia
  • MMA Methylmalonic Acidemia
  • FIG.4 depicts the differences between healthy (normal) human subjects, and subjects having a disease associated with propionate catabolism, such as propionic acidemia (PA).
  • PA propionic acidemia
  • FIG.5 depicts schematics of the major pathway (FIG.5A) and minor pathways (FIG.5B) of propionate catabolism in healthy human subjects.
  • propionyl CoA is carboxylated to D-methylmalonyl CoA by the enzyme Propionyl CoA Carboxylase (PCC), which is isomerized to L-methylmalonyl CoA.
  • PCC Propionyl CoA Carboxylase
  • MUT Methylmalonyl CoA Mutase
  • succinyl CoA which is then incorporated into the citric acid cycle.
  • FIG.5C depicts a schematic showing the metabolic relationship between PA and MMA.
  • FIG.5D depicts enzyme and other deficiencies in PA and MMA.
  • FIG.6 depicts a graph showing propionic acidemia biomarkers in
  • FIG.6A, FIG. 6B, and FIG.6C depict graphs showing detection of blood biomarkers
  • FIG.6A propionylcarnitine/acetylcarnitine ratio
  • FIG.6B propionate concentration
  • FIG.6C 2-methylcitrate
  • FIG.6D, FIG.6E, and FIG.6F depict graphs showing the detection of urine biomarkers; propionyl-glycine (FIG.6D), Tigylglycine (FIG.6E), and 2- methylcitrate (FIG.6F).
  • FIG.7A, FIG.7B, FIG.7C and FIG.7D depict bar graphs showing the levels of endogenous (FIG.7A and FIG.7B) and radiolabeled propionic acid (FIG.7C and FIG.7D) in blood, small intestine and large intestine at various time points post
  • Isotopic propionic acid is seen at very low levels in the blood, small intestine, and cecum within 30 min, indicating that
  • FIG.8 depicts potential pathways that may be engineered into the bacteria in order to consume propionic acid and/or methylmalonic acid into inert end products.
  • FIG.8A depicts a schematic of propionate catabolism, resulting in an inert product.
  • FIG.8B, FIG. 8C and FIG.8D depict schematics of three exemplary pathways, which can be utilized for propionate or methylmalonic acid catalysis.
  • the methylmalonyl-CoA (human) pathway and the 2-methylcitrate pathway produce succinate.
  • a succinate exporter can also be expressed in the engineered bacteria.
  • the methylmalonyl-CoA human pathway
  • 2-methylcitrate pathway produce succinate.
  • a succinate exporter can also be expressed in the engineered bacteria.
  • the succinate exporter can also be expressed in the engineered bacteria.
  • FIG.8D depicts a schematic showing a rearranged version of FIG.8C, showing predictions for the fate of the carbon from propionic acid.
  • the carbon is stored as PHA polymers in the cell.
  • the MMCA pathway propionate is consumed via the TCA cycle (releasing the carbon as CO2) or succinate is exported.
  • FIG.9 depicts schematics showing the activation of propionate to propionyl CoA.
  • FIG.9A shows a schematic of propionate activation through PrpE.
  • PrpE converts propionate and free CoA to propionyl-CoA in an irreversible, ATP-dependent manner, releasing AMP and PPi (pyrophosphate).
  • PrpE can be inactivated by postranslational modification of the active site lysine.
  • Protein lysine acetyltrasferase (Pka) in E. coli carries out the propionylation of PrpE.
  • the enzyme CobB depropionylates PrpE-Pr, making the inactivation reversible.
  • the genetically engineered bacteria comprise ⁇ pka to prevent inactivation of PrpE and to increase activity through the downstream catabolic pathways.
  • FIG.9B shows a schematic of propionate activation through pct. Pct converts propionate and acetyl-CoA to propionyl-CoA and acetate in a reversible reaction.
  • FIG.10 depicts a schematic of the polyhydroxyalkanoate pathway (FIG.10A) and chemical structures of the polymers produced from propionate through the PHA pathway (FIG.10B) and an exemplary circuit design for the engineered bacteria of the disclosure (FIG.10C).
  • the PHA pathway is a heterologous bacterial pathway used for carbon storage as polymers.
  • the prpE, phaB, phaC, and phaA genes are expressed under the control of an inducible promoter. PrpE, PhaB, PhaC, and PhaA are capable of catabolizing propionate or propionyl CoA into polyhydroxybutyrate, polyhydroxyvalerate, or P(HV-co- HB).
  • PrpE a propionate-CoA ligase converts propionate to propionyl CoA.
  • PhaA a beta-ketothiolase, then converts propionyl CoA to 3-keto-valeryl-CoA or converts acetyl-CoA to acetoacetyl-CoA.
  • PhaB an acetoacetyl-CoA reductase, then converts acetoacetyl-CoA into 3-hydroxy-butyryl-CoA or 3-keto-valeryl-CoA to 3-hydroxy-valeryl- CoA.
  • PhaC a polyhydroxyalkanoate synthase converts 3-hydroxy-butyryl-CoA into polyhydroxybutyrate or 3-hydroxy-valeryl-CoA to polyhydroxyvalerate or converts polyhydroxybutyrate and polyhydroxyvalerate to P(HV-co-HB).
  • the phaBCA genes are from Acinetobacter sp RA3849 and are codon-optimized for E. coli.
  • the E. coli Nissle prpE gene and the codon-optimized phaBCA genes are under the control of an aTc-inducible promoter in a single operon.
  • FIG.11 depicts a schematic of the gene organization of an exemplary construct, comprising a prpE-phaBCA gene cassette under the control of a tetracycline inducible promoter sequence, on a ⁇ 10-copy, kanamycin-resistant plasmid.
  • FIG.12 depicts a graph showing propionate concentrations over time in samples comprising genetically engineered bacteria expressing the polyhydroxyalkanoate (PHA) pathway on a ⁇ 10-copy plasmid, as compared to wild type Nissle controls, in the presence and absence of the inducer molecule.
  • Bacteria were induced with ATC (or left uninduced), and then grown in culture medium supplemented to an OD600 of 2.0.
  • Samples were harvested by centrifugation and resuspended in M9 miminal media. The activity of resuspended samples was measured by incoculating samples into M9 minimal media supplemented with glucose and sodium propionate (3 mM) to an OD600 of 1.0.
  • the graph depicts propionate consumption by the polyhydroxylalkanoate circuit design for the engineered bacteria (SYN-PHA) in the induced as compared to wild type Nissle.
  • Propionate assay was initiated with ⁇ 10 9 cfu/mlpre-induced bacteria and the propionate consumption rate was ⁇ 1.4 umol hr-1 per 10 9 cells.
  • FIG.13 depicts graphs showing propionate (FIG.13A), acetate (FIG.13B) and butyrate (FIG.13C) concentrations over time in samples comprising genetically engineered bacteria expressing the polyhydroxyalkanoate (PHA) pathway on a ⁇ 10 copy plasmid (SYN-PHA), as compared to wild type Nissle controls, both in the presence of the inducer molecule.
  • the PHA assay was performed in a mixture of short chain fatty acids to mimic the colon ratios (propionate:acetate:butyrate, approximately 6:10:4).
  • Bacteria were induced with ATC (or left uninduced), and then grown in culture medium supplemented to an OD600 of 2.0.
  • Samples were harvested by centrifugation and resuspended in M9 miminal media. The activity of resuspended samples was measured by incoculating samples into M9 minimal media supplemented with glucose and sodium propionate (6 mM), acetate (10 mM), and butyrate (4 mM) to an OD600 of 1.0. Samples were removed at 0 hrs, 1.5, 3, and 4.5 hrs post-inoculation, and propionate concentrations were determined by mass spectrometry. The data show that propionate consumption rate is consistent in the presence or absence of acetate and butyrate, and that the PHA pathway does not significantly affect acetate and butyrate concentrations.
  • FIG.14 depicts graphs showing propionate concentrations over time in samples comprising genetically engineered bacteria expressing an inducible
  • PHA polyhydroxyalkanoate
  • SYN- PHA ⁇ 10 copy plasmid
  • PHA polyhydroxyalkanoate
  • These strains were further supplemented with an second plasmid ( ⁇ 15-copies) expressing one of the genes, ie., prpE (FIG.14A), phaB (FIG.14B), phaC (FIG.14C), and phaB (FIG.14D), under the control of an inducible promoter, ie., an arabinose inducible promoter.
  • an inducible promoter ie., an arabinose inducible promoter.
  • the genetically engineered bacteria comprise a prpE-phaBCA operon, in which PhaC levels are increased through the utilization of a strong ribosome binding site (RBS).
  • the genetically engineered bacteria comprising a prpE-phaBCA operon further comprise an additional copy of phaC.
  • FIG.15 depicts schematics of the methylmalonyl-CoA pathway and exemplary methylmalonylCoA circuit designs.
  • FIG.15A depicts a schematic showing PrpE reaction and by the methylmalonylCoA pathway, in which the products of the prpE, pccB, accA1, mmcE, mutA, and mutB genes convert propionate into succinate, and which can be used for circuit design.
  • the methylmalonyl-CoA pathway carries out reactions homologous to those in the mammalian pathway and the pathway is assembled from heterologous bacterial enzymes.
  • genes accA from Streptomyces coelicolor
  • pccB from Streptomyces coelicolor
  • mmcE from Propionibacterium freudenreichii
  • mutAB from Propionibacterium freudenreichii
  • FIG.15B depicts a schematic showing an exemplary circuit design of the disclosure, in which the genetically engineered bacteria comprise a gene cassette comprising the prpE, pccB, accA1, mmcE, mutA, and mutB genes under the control of an inducible promoter, e.g., a aTc-inducible promoter.
  • an inducible promoter e.g., a aTc-inducible promoter.
  • FIG.15C depicts depicts a schematic showing an exemplary circuit design of the disclosure, in which the genetically engineered bacteria comprise a cassette comprising prpE, pccB, accA1, under the control of a first inducible promoter, e.g., Ptet (aTc inducible) and a second cassette comprisige mmcE and mutAB under the control of a second inducible promoter, e.g., Para (arabinose inducible). Induction of the pathway requires the addition of aTc and arabinose.
  • a succinate exporter may also be expressed in the engineered bacteria.
  • FIG.16 depicts schematics of the gene organization of exemplary constructs.
  • FIG.16A depicts a schematic of the gene organization of an exemplary construct, comprising a mmcE-mutA-mutB gene cassette under the control of an arabinose inducible promoter sequence, on a ⁇ 15-copy, ampicillin-resistant plasmid.
  • FIG.16B depicts a schematic of the gene organization of an exemplary construct, comprising a prpE-accA-pccB gene cassette under the control of a tetracycline inducible promoter sequence, on a ⁇ 10-copy, kanamycin-resistant plasmid.
  • FIG.17 depicts schematics of the MMCA pathway combined with a succinate exporter and related exemplary genetic circuits and synthetic biotics.
  • FIG.17A depicts a schematic of propionate and/or methylmalonic acid catabolism through the MMCA pathway.
  • the resulting succinate can be metabolized through the TCA cycle or removed from the bacterial cell through an exporter.
  • Exemplary exporters include sucE1 succinate exporter (e.g., from Corynebacterium glutamicum) and/or the native Nissle succinate exporter dcuC.
  • FIG.17B depicts an exemplary circuit or gene cassette for the expression of the sucE1 succinate exporter (e.g., from Corynebacterium glutamicum) under the control of an inducible promoter, e.g., an arabinose-inducible promoter.
  • This construct can either be expressed in the synthetic biotic on a plasmid, or it can be integrated into the genome.
  • a knock-in of the construct which deletes the araBA genes and part of the araD gene, can be performed, which elimitates metabolism of arabinose by E. coli.
  • FIG.17C depicts a schematic of an exemplary synthetic biotic of the disclosure comprising a gene cassette expressing the prpE, phaB, phaC, and phaA genes under the control of an inducible promoter.
  • the synthetic biotic further comprises a gene cassette expressing the sucE1 gene under the control of an inducible promoter.
  • FIG.17D depicts a schematic of a construct comprising the sucE1 succinate exporter (from Corynebacterium glutamicum.
  • FIG.17E depicts a schematic of a construct comprising the E. coli dcuC succinate transporter.
  • FIG. 17F depicts a schematic of a construct comprising or comprising both sucE1 and dcuC transporters.
  • FIG.18 depicts a graph showing propionate concentrations over time in samples comprising genetically engineered bacteria expressing the methylmalonyl-CoA pathway circuit (SYN-MMCA) or a polyhydroxylalkanoate pathway circuit (SYN-PHA) on a ⁇ 10- and ⁇ 15-copy plasmids as compared to wild type Nissle controls, in the presence of the inducer molecule.
  • Bacteria were induced ATC or ATC and arabinose (or left uninduced), and then grown in culture medium supplemented to an OD600 of 2.0. Samples were harvested by centrifugation and resuspended in M9 miminal media.
  • the activity of resuspended samples was measured by incoculating samples into M9 minimal media supplemented with glucose and sodium propionate (3 mM) to an OD600 of 1.0. Samples were removed at were removed at 0 hrs, 1.5, 3, 4.5, and 18 hrs post-inoculation, cells were removed, and propionate concentrations were determined by mass spectrometry.
  • the graph depicts propionate consumption by the methylmalonyl-CoA pathway or a polyhydroxylalkanoate circuit design for the engineered bacteria in the induced as compared to wild type Nissle. Propionate assay was initiated with ⁇ 109 cfu/mlpre-induced bacteria and the propionate consumption rate was ⁇ 3.8 ⁇ mol/hr/10 9 bacteria in the strain expressing the methylmalonyl-CoA pathway circuit.
  • FIG.19 depicts one example of a normal pathway for the catabolism of propionate via the methylcitrate cycle in bacteria, for example, E. coli.
  • PrpE a Propionate-CoA ligase
  • PrpC a 2-methylcitrate synthetase
  • PrpD a 2-methylcitrate dehydrogenase
  • PrpB a 2- methylisocitrate lyase
  • FIG.20 depicts schematics of the 2-methylcitrate cycle in bacteria, e.g., E. coli, (FIG.20A) and a schematice of an exemplary circuit design for the engineered bacteria (FIG.20B).
  • the prpB, prpC, prpD, and prpE genes are expressed under the control of an inducible promoter in order to produce succinate and pyruvate.
  • a succinate exporter may also be expressed in the engineered bacteria.
  • 20C depicts a schematic of the gene organization of an exemplary construct, comprising a prpBCDE gene cassette under the control of a tetracycline inducible promoter sequence, on a ⁇ 10-copy, kanamycin-resistant plasmid.
  • FIG.21 depicts schematics of exemplary synthetic biotics of the disclosure for the treatment of propionic acidemia and/or methylmalonic acidemia and/or disorders characterized by propionic acidemia and/or methylmalonic acidemia.
  • FIG.21A depicts a schematic of an exemplary synthetic biotic of the disclosure comprising a gene cassette expressing the prpE, phaB, phaC, and phaA genes under the control of an inducible promoter. PrpE, PhaB, PhaC, and PhaA are capable of catabolizing propionate or propionyl CoA and/or methylmalonic acid or methylmalonyl CoA into P(HV-co-HB).
  • FIG.21B depicts a schematic of a synthetic biotic of FIG.1A or FIG 21A, with the addition of a ThyA auxotrophy.
  • FIG. 21C depicts the synthetic biotic of FIG.1B, with the additiona of a ThyA auxotrophy.
  • FIG. 21D depicts a schematic of an exemplary synthetic biotic of the disclosure comprising a gene cassette expressing prpE, accA, pccB, mmcE, mutA and mutB as two polycistronic messages from two inducible promoters. PrpE, accA, pccB, mmcE, mutA and mutB are capable of catabolizing propionate or propionyl CoA and/or methylmalonic acid or methylmalonyl CoA into succinate, which can be utilized through the TCA cycle or exported from the cell.
  • FIG.21E depicts a schematic of a synthetic biotic comprising one or more of two different gene cassettes for propionate catabolism (PHA and MMCA pathway cassettes).
  • FIG.21F depicts a schematic of an exemplary synthetic biotic of the disclosure comprising a gene cassette expressing prpE, accA, pccB, mmcE, mutA and mutB as two polycistronic messages from two inducible promoters in combination with MatB.
  • FIG.21G depicts a schematic of a synthetic biotic comprising one or more of two different gene cassettes for propionate catabolism (PHA and MMCA pathway cassettes) in combination with MatB.
  • FIG.22 depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter
  • FIG.22 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
  • 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.
  • 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.23 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.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • 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.
  • 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.24 depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • 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.
  • TetR Tet repressor
  • 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).
  • araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.
  • FIG.25 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
  • FIG.26 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
  • FIG.27 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.
  • 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.
  • recombinases can be used to further control the timing of cell death.
  • FIG.28 depicts a schematic of one non-limiting embodiment of the disclosure, in which the genetically engineered bacteria produces equal amount of a Hok toxin and a short-lived Sok anti-toxin.
  • the cell loses the plasmid, the anti-toxin decays, and the cell dies.
  • the cell produces equal amounts of toxin and anti-toxin and is stable.
  • the center panel the cell loses the plasmid and anti-toxin begins to decay.
  • the anti-toxin decays completely, and the cell dies.
  • FIG.29 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.
  • FIG.30 depicts an example of a genetically engineered bacteria that comprises a plasmid that has been modified to create a host-plasmid mutual dependency, such as the GeneGuard system described in more detail herein.
  • FIG.31 depicts an exemplary schematic of the E. coli 1917 Nissle
  • chromosome comprising multiple mechanisms of action (MoAs).
  • a single synthetic biotic may have multiple mechanisms of action (MOAs) based on the insertion of multiple copies of the same synthetic circuit or the insertion of different synthetic circuits at different sites in a bacterial chromosome.
  • FIG.32 depicts a map of integration sites within the E. coli Nissle
  • chromosome chromosomes. 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.
  • FIG.33 depicts three bacterial strains which constitutively express red fluorescent protein (RFP).
  • RFP red fluorescent protein
  • FIG.34 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.
  • FIG.35 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.
  • FIG.36 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.
  • FIG.37 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.
  • 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.
  • FIG.38 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.
  • HlyB an ATP- binding cassette transporter
  • HlyD a membrane fusion protein
  • TolC an outer membrane protein
  • FIG.39 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.
  • FIG.40 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
  • 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).
  • FIG.41 depicts ⁇ -galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters.
  • 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 (+O2) or anaerobic conditions (-O2). Samples were removed at 4 hrs and the promoter activity based on ⁇ -galactosidase levels was analyzed by performing standard ⁇ -galactosidase colorimetric assays.
  • FIG.42 depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P fnrS ).
  • LacZ encodes the ⁇ -galactosidase enzyme and is a common reporter gene in bacteria.
  • FIG.42B depicts FNR promoter activity as a function of ⁇ -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 ⁇ -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.42C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.
  • FIG.43 depicts ATC (FIG.43A) or nitric oxide-inducible (FIG.43B) reporter constructs. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible 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.43C depicts a schematic of the constructs.
  • FIG.43D 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).
  • FIG.44 depicts the prpR propionate-responsive inducible promoter.
  • the sequence for one propionate-responsive promoter is also disclosed herein as (SEQ ID NO:
  • FIG.45 depicts a schematic of a wild-type clbA construct and a clbA knock- out construct.
  • FIG.46 depicts a schematic of non-limiting processes for designing and producing the genetically engineered bacteria of the present disclosure.
  • the step of “defining” comprises 1. Identification of diverse candidate approaches based on microbial physiology and disease biology; 2. Use of bioinformatics to determine candidate metabolic pathways; the use of prospective tools to determine performance targets required of optimized engineered synthetic biotics.
  • the step of“designing” comprises the use of 1.
  • the step of“Building” comprises 1. Building core structures“chassies” 2. Stably integrating engineered circuits into optimal chromosomal locations for efficient expression; 3. Employing unique functional assays to assess genetic circuit fidelity and activity.
  • the step of“integrating” comprises 1. Use of chromosomal markers, which enable monitoring of synthetic biotic localization and transit times in animal models; 2. Leveraging expert microbiome network and bioinformatics support to expand understanding of how specific disease states affect GI microbial flora and the behaviors of synthetic biotics in that environment; 3. Activating process development research and optimization in-house during the discovery phase, enabling rapid and seamless transition of development candidates to pre-clinical progression; Drawing upon extensive experience in specialized disease animal model refinement, which supports prudent, high quality testing of candidate synthetic biotics.
  • FIG.47A, FIG.47B, FIG.47C, FIG.47D, and FIG.47E depict a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure.
  • FIG.47A depicts the parameters for starter culture 1 (SC1): loop full– glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm.
  • FIG.47B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C, shaking at 250 rpm.
  • FIG.47C 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.
  • FIG.47D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash 1X 10% glycerol/PBS, centrifugation, re- suspension 10% glycerol/PBS.
  • FIG.47E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.
  • the present disclosure provides engineered bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with propionate catabolism, such as propionic acidemia, methylmalonic acidemia, or vitamin B 12 deficiency.
  • the engineered bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, at least one propionate catabolism enzyme.
  • 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.
  • engineered bacterial cell refers to a bacterial cell or bacteria that have been genetically modified from their native state.
  • an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and/or 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.
  • engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.
  • a“recombinant microorganism” refers to a microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state.
  • a“recombinant bacterial cell” or“recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state.
  • 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.
  • recombinant bacterial cells may comprise exogenous nucleotide sequences stably
  • A“programmed microorganism” or“engineered microorganism” refers to a microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state to perform a specific function, e.g., to metabolize propionate and/or one or more of its metabolites.
  • 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.
  • A“programmed bacterial cell” or“engineered bacterial cell” is a bacterial cell that has been genetically modified from its native state.
  • 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 metabolize a propionate and/or one or more of its metabolites.
  • 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.
  • 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.
  • the term“gene” refers to any nucleic acid sequencethat 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.
  • 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.
  • 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.
  • 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.
  • a“heterologous” gene or“heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature.
  • 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.
  • 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.
  • 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.
  • the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism.
  • the term“transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.
  • 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.
  • the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013).
  • the non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette.
  • “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.
  • 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.
  • 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 a propionate catabolism enzyme.
  • 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 propionate catabolism enzyme.
  • 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.
  • the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter or other promoter disclosed 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 a propionate 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.
  • the stable bacterium may be a genetically engineered bacterium comprising a gene encoding a propionate catabolism enzyme, in which the plasmid or chromosome carrying the gene is stably maintained in the bacterium, such that propionate catabolism enzyme can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo.
  • 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.
  • a“gene cassette” or“operon” encoding a propionate catabolism pathway refers to the two or more genes that are required to catabolize propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA into an inert end-product, e.g., succinate or polyhydroxyalkanotes.
  • 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.
  • 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.
  • 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.
  • “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.
  • operably linked refers to a nucleic acid sequence, e.g., a gene encoding a propionate 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 propionate catabolism enzyme.
  • the regulatory sequence acts in cis.
  • a gene may be“directly linked” to a regulatory sequence in a manner which allows expression of the gene.
  • a gene may be “indirectly linked” to a regulatory sequence in a manner which allows expression of the gene.
  • 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.
  • 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.
  • Constant 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 ⁇ S 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 ⁇ 32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli ⁇ 70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E.
  • a constitutive Escherichia coli ⁇ S promoter e.g., an o
  • coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110
  • BBa_M13110 Bacillus subtilis ⁇ A promoter
  • promoter veg a constitutive Bacillus subtilis ⁇ A promoter
  • BBa_K823002 P veg (BBa_K823003)
  • a constitutive Bacillus subtilis ⁇ B promoter e.g., promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)
  • a Salmonella promoter e.g., Pspv2 from Salmonella (BBa_K112706), Pspv from Salmonella (BBa_K112707)
  • a bacteriophage T7 promoter e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BB
  • 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.
  • 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.
  • oxygen level-dependent promoters e.g., FNR-inducible promoter
  • RNS inflammatory response
  • ROS ROS promoters
  • inducible promoters include, but are not limited to, an FNR responsive promoter, a P araC promoter, a P araBAD promoter, and a P TetR promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.
  • 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.
  • 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).
  • 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.
  • genetic modification 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 a propionate 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.
  • 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.
  • the term“genetic modification that increases import of propionate into the bacterial cell” refers to a genetic modification that increases the uptake rate or increases the uptake quantity of propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA or metabolites thereof, into the cytosol of the bacterial cell, as compared to the uptake rate or uptake quantity of the propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA into the cytosol of a bacterial cell not having said modification, e.g., a wild-type bacterial cell.
  • a engineered bacterial cell having a genetic modification that increases import of propionate into the bacterial cell refers to a bacterial cell comprising a heterologous gene encoding a transporter of propionate.
  • a recombinant bacterial cell having a genetic modification that increases import of propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites from the bacterial cell comprises a genetic mutation in a native gene.
  • a recombinant bacterial cell having a genetic modification that increases import of a propionate and/or its metabolites from the bacterial cell comprises a genetic mutation in a native promoter, which increases or activates transcription of the gene which increases import of propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites.
  • a recombinant bacterial cell having a genetic modification that increases import of p propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites from the bacterial cell comprises a genetic mutation leading to overexpression of a activator of an importer (transporter) of propionate and/or its metabolites.
  • a recombinant bacterial cell having a genetic modification that increases import of propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites from the bacterial cell comprises a genetic mutation which increases or activates translation of the gene encoding the transporter (importer).
  • the term“genetic modification that increases import of a propionate and/or its metabolites into the bacterial cell” refers to a genetic modification that increases the uptake rate or increases the uptake quantity of a propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites into the cytosol of the bacterial cell, as compared to the uptake rate or uptake quantity of propionate and/or its metabolites into the cytosol of a bacterial cell not having said modification, e.g., a wild-type bacterial cell.
  • an engineered bacterial cell having a genetic modification that increases import of propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites 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 propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA and/or their metabolites.
  • the genetically engineered bacteria comprising genetic modification that increases import of propionate and one or more of its metabolites into the bacterial cell comprise gene sequence(s) encoding a propionate transporter or other amino acid transporter that transports one or more propionate metabolites into the bacterial cell, for example a transporter that is capable of transporting methylmalonic acid into a bacterial cell.
  • the transporter can be any transporter that assists or allows import of propionate and/or metabolites thereof into the cell.
  • the propionate transporter is one of MctC, PutP_6, or any other propionate transporters described herein.
  • the engineered bacterial cell contains gene sequences encoding MctC, PutP_6, or any other propionate transporters described herein. In some embodiments, the engineered bacteria comprise more than one copy of gene sequence encoding a propionate transporter. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding more than one propionate transporter, e.g., two or more different propionate transporters.
  • propionate refers to C2H5COO-. Propionate is the conjugate base of propionic acid.
  • Propionyl CoA Carboxylase PCC
  • vitamin B7 biotin
  • L-methylmalonyl CoA L-methylmalonyl CoA
  • MUT Methylmalonyl CoA Mutase
  • propionate binding protein refers to a protein which can bind to propionate and/or one or more propionate metabolites, including, but not limited to, methylmalonate and/or methylmalonic acid.
  • 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.
  • propionate transporter refers to a polypeptide which functions to transport propionate and/or one or more of its metabolites, including, but not limited to, methylmalonate and/or methylmalonic acid into the bacterial cell.
  • the phrase“propionate and/or its metabolites” or“propionate and/or one or more of its metabolites”, includes any metabolite of propionate, such as any of the metabolites described herein, and also includes propionyl CoA, methylmalonic acid, or methylmalonyl CoA.
  • “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.
  • GI gastrointestinal
  • 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.
  • 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;
  • Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.
  • 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.
  • 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 propionate e.g., Propionic Acidemia (PA) or Methylmalonic Acidemia (MMA), may be caused by inborn genetic mutations for which there are no known cures. Diseases can also be secondary to other conditions, e.g., liver diseases.
  • Treating diseases involving the catabolism of propionate may encompass reducing normal levels of propionate, propionic acid, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA, reducing excess levels of propionate, propionic acid, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA, or eliminating propionate, propionic acid, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA, and does not necessarily encompass the elimination of the underlying disease.
  • the term“catabolism” refers to the conversion of an odd-chain fatty acid, cholesterol, or branched chain amino acid, such as methionine, threonine, isoleucine, or valine, into its corresponding propionyl CoA, methylmalonyl CoA, or succinyl CoA.
  • “abnormal catabolism” refers to a decrease in the rate or the level of conversion of an odd-chain fatty acid, cholesterol, or branched chain amino acid into its corresponding propionyl CoA, methylmalonyl CoA, or succinyl CoA, leading to the build-up of propionyl CoA or methylmalonyl CoA in the blood or the brain of a subject.
  • build-up of propionyl CoA or methylmalonyl CoA in the blood or the brain of a subject becomes toxic and leads to the development of a disease or disorder associated with the abnormal catabolism of propionate in the subject.
  • “Catabolism” e.g.,“Propionate catabolism” also refers to the breakdown of propionate and/or methylmalonic acid to one or more of its breakdown products as described herein.
  • a“disorder involving the catabolism of propionate” is a disease or disorder involving the abnormal catabolism of propionate, propionyl CoA, methylmalonic acid, or methylmalonyl CoA.
  • the term“disorder involving the abnormal catabolism of propionate” refers to a disease or disorder wherein the catabolism of propionate, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA is abnormal.
  • “abnormal catabolism” refers to a decrease in the rate or the level of conversion of propionyl CoA into methylmalonyl CoA, or a decrease in the rate or the level of conversion of methylmalonyl CoA into succinyl CoA, leading to the build-up of propionate, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA in the blood or the brain of a subject.
  • build-up of the propionate, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA in the blood or the brain of a subject becomes toxic and leads to the development of a disease or disorder associated with the abnormal catabolism of propionate in the subject.
  • the disorder involving the abnormal catabolism of propionate is Propionic Acidemia or Methylmalonic Acidemia.
  • 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.
  • 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.
  • “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.
  • the exogenous environmental conditions are specific to the gut of a mammal.
  • the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the small intestine of a mammal.
  • the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut.
  • exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate.
  • the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s).
  • the exogenous environmental condition is specific to a propionate catabolism enzyme disease, e.g., Propionic Acidemia and/or Methylmalonic Acidemia.
  • the exogenous environmental condition is a low-pH environment.
  • the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter.
  • the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter.
  • 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.
  • oxygen level-dependent transcription factors include, but are not limited to, FNR (fumarate and nitrate reductase), ANR, and DNR.
  • FNR fluarate and nitrate reductase
  • ANR anaerobic nitrate respiration
  • DNR dissimilatory nitrate respiration regulator
  • a promoter 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
  • FNR transcriptional regulator
  • 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).
  • exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut.
  • 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.
  • the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter.
  • 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.
  • the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example,
  • the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure.
  • 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).
  • the loss of exposure to an exogenous environmental condition inhibits the activity of an inducible promoter, as the exogenous
  • “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.
  • 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.
  • Microorganism refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, and protozoa.
  • the microorganism is engineered (“engineered microorganism”) to produce one or more therpauetic molecules, e.g., lysosomal enzyme(s).
  • the engineered microorganism is an engineered bacterium.
  • the engineered microorganism is an engineered virus.
  • Non-pathogenic bacteria refer to bacteria that are not capable of causing disease or harmful responses in a host.
  • non-pathogenic bacteria are Gram-negative bacteria.
  • non-pathogenic bacteria are Gram-positive bacteria.
  • non-pathogenic bacteria do not contain lipopolysaccharides (LPS).
  • LPS lipopolysaccharides
  • 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,
  • Bacillus coagulans Bacillus subtilis
  • Bacteroides fragilis Bacteroides subtilis
  • Non- pathogenic bacteria also include commensal bacteria, which are present in the indigenous microbiota of the gut.
  • the disclosure further includes non-pathogenic Saccharomyces, such as Saccharomyces boulardii.
  • Naturally pathogenic bacteria may be genetically engineered to reduce or eliminate pathogenicity.
  • 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.
  • the host organism is a mammal.
  • the host organism is a human.
  • the probiotic bacteria are Gram-negative bacteria.
  • the probiotic bacteria are Gram- positive bacteria.
  • probiotic bacteria examples include, but are not limited to, certain strains belonging to the genus Bifidobacteria, Escherichia Coli, Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus,
  • Lactobacillus paracasei and Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No.5,589,168; U.S. Patent No.6,203,797; U.S. Patent 6,835,376).
  • the probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
  • Non- pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • 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.
  • 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).
  • 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),
  • “modulate” and“treat” refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition.
  • “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.
  • Those in need of treatment may include individuals already having a particular medical 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 propionate and/or one or more of its metabolites e.g., Propionic Acidemia and/or Methylmalonic Acidemia, may be caused by inborn genetic mutations for which there are no known cures. Diseases can also be secondary to other conditions.
  • Treating diseases involving the catabolism of propionate and methylmalonate may encompass reducing normal levels of of propionate and/or one or more of its metabolites, reducing excess levels of of propionate and/or one or more of its metabolites, or eliminating of propionate and/or one or more of its metabolites and does not necessarily encompass the elimination of the underlying disease.
  • the payload refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus.
  • the payload is a therapeutic payload, e.g., a propionate catabolic enzyme or a propionate transporter polypeptide.
  • the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR.
  • the payload comprises a regulatory element, such as a promoter or a repressor.
  • the payload comprises an inducible promoter, such as from FNRS.
  • the payload comprises a repressor element, such as a kill switch.
  • the payload comprises an antibiotic resistance gene or genes.
  • the payload is encoded by a gene, multiple genes, gene cassette, or an operon.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism.
  • the genetically engineered microorganism comprises two or more payloads.
  • 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).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • polypeptides “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Polypeptides also include fusion proteins.
  • the term“variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide.
  • 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
  • “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
  • amino acids belonging to one of the following groups represent conservative changes or
  • substitutions -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.
  • 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.
  • 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.
  • 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) may be naturally occurring variants as well as artificially designed ones.
  • 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.
  • 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.
  • 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.
  • 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.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • 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.
  • the polypeptide to be secreted include a“secretion tag” of either RNA or peptide origin to direct the polypeptide to specific secretion systems.
  • the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria.
  • 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.
  • 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.
  • 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.
  • 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.
  • the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl.
  • 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.
  • a "pharmaceutical composition” refers to a preparation of bacterial cells with other components such as a physiologically suitable carrier and/or excipient.
  • 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.
  • 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.
  • 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.
  • 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 the disease.
  • therapeutically effective amount as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.
  • bacteriostatic or “cytostatic” refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division,
  • bactericidal refers to a molecule or protein which is capable of killing the engineered bacterial cell of the disclosure.
  • 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.
  • anti-toxin or“antitoxin,” as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin.
  • anti- toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.
  • 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.
  • “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.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • 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.
  • the disclosure provides a bacterial cell that comprises at least one
  • 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.
  • 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.
  • a Bacteroides fragilis Bacteroides thetaiotaomicron
  • Bacteroides subtilis Bacteroides subtilis
  • Bifidobacterium animalis Bifidobacterium bifidum
  • Bifidobacterium infantis Bifidobacterium lactis
  • 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
  • the bacterial cell is a Bifidobacterium infantis bacterial cell.
  • 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
  • the bacterial cell is a Oxalobacter formigenes bacterial cell. In another embodiment, the bacterial cell does not include Oxalobacter formigenes.
  • the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.
  • 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 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), and E. coli Nissle does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not
  • 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 al., 2007).
  • the engineered bacterial cell does not colonize the subject.
  • 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.
  • the bacterial cell is a genetically engineered bacterial cell.
  • the bacterial cell is an engineered bacterial cell.
  • the disclosure comprises a colony of bacterial cells.
  • the disclosure provides an engineered bacterial culture which comprises engineered bacterial cells.
  • 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.
  • 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.
  • the genetically engineered bacteria is an auxotroph or a conditional auxotroph.
  • 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 thi1 auxotroph.
  • the engineered bacteria have more than one auxotrophy, for example, they may be a ⁇ thyA and ⁇ dapA auxotroph.
  • the genetically engineered bacteria further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein.
  • 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.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • 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.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • 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 P araBAD .
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the genetically engineered bacteria is an auxotroph and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.
  • 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.
  • 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.
  • the disclosure provides an engineered bacterial culture which reduces levels of propionate, propionyl CoA, and/or methylmalonyl CoA in the media of the culture. In one embodiment, the levels of the propionate, propionyl CoA, and/or
  • methylmalonyl CoA are reduced by about 50%, about 75%, or about 100% in the media of the cell culture.
  • the levels of the propionate, propionyl CoA, and/or methylmalonyl CoA 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.
  • the levels of the propionate, propionyl CoA, and/or methylmalonyl CoA are reduced below the limit of detection in the media of the cell culture.
  • 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 propionate and/or metabolizing one or more propionate metabolite(s).
  • enzymes and propionate metabolic pathways are described herein.
  • propionate metabolic pathways include, but are not limited to, one or more of the polyhydroxyalkanoate (PHA), methylmalonyl-CoA (MMCA), and 2-methylcitrate (2MC) pathways, e.g., as described herein.
  • the disclosure provides a bacterial cell that comprises one or more heterologous gene sequence(s) and/or gene cassette(s) encoding one or more propionate catabolism enzyme(s) or other protein(s) that results in a decrease in levels of propionate and/or certain propionate metabolites, e.g., methylmalonate.
  • 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
  • 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
  • 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.
  • the genetically engineered bacteria are selected from the group consisting of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei,
  • the genetically engineered bacteria are selected from Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis,
  • 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
  • the bacterial cell is a
  • 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.
  • 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).
  • 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
  • genes from one or more different species can be introduced into one another, e.g., the phaBCA genes from Acinetobacter sp RA3849, the accA gene from Streptopmyces coelicolor, pccB gene from Streptopmyces coelicolor, mmcE gene from Propionibcterium freudenreichii or the mutAB genes from Propionibcterium freudenheimii, or matB, derived from Rhodopseudomonas palustris, can be expressed in Escherichia coli.
  • the genes are codon optimized, e.g., for expression in E. coli.
  • the recombinant bacterial cell does not colonize the subject having the disorder.
  • Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009).
  • the residence time is calculated for a human subject.
  • residence time in vivo is calculated for the genetically engineered bacteria of the invention.
  • 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.
  • the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein.
  • the disclosure provides a recombinant bacterial culture which reduces levels of propionate in the media of the culture.
  • the levels of propionate and/or one or more of its metabolites are reduced by about 50%, about 75%, or about 100% in the media of the cell culture.
  • the levels of propionate and/or one or more of its metabolites 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.
  • the levels of propionate and/or one or more of its metabolites are reduced below the limit of detection in the media of the cell culture.
  • the gene encoding a propionate 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.
  • the gene encoding a propionate 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.
  • the gene encoding a propionate 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.
  • the gene encoding a propionate 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.
  • the genetically engineered bacteria comprising gene sequence encoding a propionate catabolism enzyme is an auxotroph.
  • 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 thi1 auxotroph.
  • the engineered bacteria have more than one auxotrophy, for example, they may be a ⁇ thyA and ⁇ dapA auxotroph.
  • the genetically engineered bacteria comprising gene sequence encoding a propionate catabolism enzyme lacks functional ilvC gene sequence, e.g., is a ilvC auxotroph.
  • IlvC encodes keto acid reductoisomerase, which enzyme is required for propionate synthesis.
  • Knock out of ilvC creates an auxotroph and requires the bacterial cell to import isoleucin and valine to survive.
  • the genetically engineered bacteria comprising gene sequence encoding a propionate catabolism enzyme further comprise gene sequence(s) encoding a propionate transporter into the bacterial cell.
  • the propionate transporter is MctC, PutP_6, or any other propionate transporters described herein.
  • the bacterial cell contains gene sequence encoding MctC, PutP_6, or any other propionate transporters described herein.
  • the genetically engineered bacteria comprising gene sequence encoding a propionate 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.
  • the genetically engineered bacteria comprising gene sequence encoding a propionate catabolism enzyme further comprise gene sequence(s) encoding one or more antibiotic gene(s), such as any of the antibiotic genes disclosed herein.
  • the genetically engineered bacteria comprising a propionate catabolism enzyme further comprise a kill-switch circuit, such as any of the kill- switch circuits provided herein.
  • the genetically engineered bacteria further comprise one or more genes encoding one or more
  • 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.
  • 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.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the genetically engineered bacteria is an auxotroph comprising gene sequence encoding a propionate catabolism enzyme and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.
  • the gene encoding a propionate catabolism enzyme is present on a plasmid in the bacterium. In some embodiments, the gene encoding a propionate catabolism enzyme is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding a propionate transporter, e.g., MctC, PutP_6, or any other propionate transporters described herein, is present on a plasmid in the bacterium.
  • a propionate transporter e.g., MctC, PutP_6, or any other propionate transporters described herein
  • the gene sequence(s) encoding a propionate transporter e.g., MctC, PutP_6, or any other propionate transporters described herein, is present in the bacterial chromosome.
  • the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule is present on a plasmid in the bacterium.
  • 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.
  • the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium.
  • the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial
  • the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene encoding the propionate catabolism enzyme such that the propionate 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.
  • bacterial cell comprises two or more distinct propionate catabolism enzymes.
  • the genetically engineered bacteria comprise multiple copies of the same propionate catabolism enzyme gene.
  • the genetically engineered bacteria comprise multiple copies of different propionate catabolism enzyme genes.
  • the gene encoding the propionate catabolism enzyme is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the propionate 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 gene encoding the propionate catabolism enzyme is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the propionate 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 gene encoding the propionate catabolism enzyme is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.
  • the bacterial cell comprises a stably maintained plasmid or chromosome carrying the at least one gene encoding a transporter of propionate and/or one or more metatabolites thereof, 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.
  • bacterial cell comprises two or more distinct copies of the at least one gene encoding a propionate transporter.
  • the genetically engineered bacteria comprise multiple copies of the same at least one gene encoding a propionate transporter.
  • the at least one gene encoding a transporter of propionate 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 propionate 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 propionate transporter, is present on a chromosome and operably linked to a directly or indirectly inducible promoter.
  • the at least one gene encoding a propionate transporter is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions.
  • the at least one gene encoding a transporter propionate and/or methylmalonate is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.
  • the promoter that is operably linked to the gene encoding the propionate catabolism enzyme and the promoter that is operably linked to the gene encoding the propionate transporter is directly induced by exogenous environmental conditions.
  • the promoter that is operably linked to the gene encoding the propionate catabolism enzyme and the promoter that is operably linked to the gene encoding the propionate transporter is indirectly induced by exogenous environmental conditions.
  • the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal.
  • the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal.
  • the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut.
  • the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal, e.g., propionate. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the bacterial cell.
  • the bacterial cell comprises a stably maintained plasmid or chromosome carrying the at least one gene encoding a propionate binding protein, such that the propionate binding protein, 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.
  • bacterial cell comprises two or more distinct copies of the at least one gene encoding a propionate binding protein.
  • the genetically engineered bacteria comprise multiple copies of the same at least one gene encoding a propionate binding protein.
  • the at least one gene encoding a propionate binding protein 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 propionate binding protein, 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 propionate binding protein, is present on a chromosome and operably linked to a directly or indirectly inducible promoter.
  • the at least one gene encoding a propionate binding protein 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 propionate binding protein, is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose. [0152] In some embodiments, the promoter that is operably linked to the gene encoding the propionate catabolism enzyme and the promoter that is operably linked to the gene encoding the propionate binding protein, is directly induced by exogenous
  • the promoter that is operably linked to the gene encoding the propionate catabolism enzyme and the promoter that is operably linked to the gene encoding the propionate binding protein is indirectly induced by exogenous environmental conditions.
  • the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal.
  • the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal.
  • the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut.
  • the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal, e.g., propionate. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell.
  • the bacterial cell comprises a gene encoding a propionate 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 propionate transporter 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 propionate binding protein is expressed under the control of the fumarate and nitrate reductase regulator (FNR) promoter.
  • FNR fumarate and nitrate reductase regulator
  • FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive.
  • FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.
  • the FNR responsive promoter comprises SEQ ID NO: 1. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 2. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 3. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 4. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 5. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 6. In yet 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: 9. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 10.
  • the FNR responsive promoter comprises SEQ ID NO: 11. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 12. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 13. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 14. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 15. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 16. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 17. [0156] In other embodiments, the FNR responsive promoter has at least about 80% identity with a nucleic acid sequence encoding any of SEQ ID NOs:1-17.
  • the FNR responsive promoter has at least about 85% identity with a nucleic acid sequence encoding any of SEQ ID NOs:1-17. In other embodiments, the FNR responsive promoter has at least about 90% identity with a nucleic acid sequence encoding any of SEQ ID NOs:1-17. In other embodiments, the FNR responsive promoter has at least about 95% identity with a nucleic acid sequence encoding any of SEQ ID NOs:1-17. 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:1-17.
  • 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:1-43.
  • multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria.
  • the genetically engineered bacteria comprise a gene encoding a propionate 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).
  • the genetically engineered bacteria comprise at least one gene encoding a propionate 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 al., 1997).
  • the genetically engineered bacteria comprise at least one gene encoding a propionate binding protein 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).
  • an alternate oxygen level-dependent promoter e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997.
  • catabolism of propionate and/or its metabolites is particularly activated in a low-oxygen or anaerobic environment, such as in the gut.
  • gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability.
  • the mammalian gut is a human mammalian gut.
  • the bacterial cell 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 gene encoding the propionate catabolism enzyme, and/or the at least one gene encoding a propionate transporter, and/or the at least one gene encoding a propionate binding protein in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions.
  • the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011).
  • the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity.
  • the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.
  • the genetically engineered bacteria comprise a wild- type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype.
  • the mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the propionate catabolism enzyme, and/or the at least one gene encoding a propionate transporter and/or the at least one gene encoding a propionate binding protein in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions.
  • the genetically engineered bacteria comprise a wild- type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and
  • 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 al., 2006).
  • the bacterial cells disclosed herein comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene.
  • the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the propionate catabolism enzyme are present on different plasmids.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the propionate catabolism enzyme and/or the at least one gene encoding a propionate transporter and/or the at least one gene encoding a propionate binding protein are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the propionate catabolism enzyme and/or the at least one gene encoding a transporter of a propionate and/or the at least one gene encoding a propionate binding protein are present on the same plasmid.
  • the transcriptional regulator is present on a chromosome.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the gene encoding the propionate catabolism enzyme and/or the at least one gene encoding a propionate transporter and/or the at least one gene encoding a propionate binding protein are present on different chromosomes.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the propionate catabolism enzyme and/or the at least one gene encoding a propionate transporter and/or the at least one gene encoding a propionate binding protein are present on the same chromosome.
  • the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability.
  • expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the propionate catabolism enzyme and/or the transporter of propionate and /or metabolites thereof and/or the propionate binding protein.
  • expression of the transcriptional regulator is controlled by the same promoter that controls expression of the propionate catabolism enzyme and/or the transporter of propionate and /or metabolites thereof, and/or the propionate binding protein.
  • the transcriptional regulator and the propionate catabolism enzyme are divergently transcribed from a promoter region.
  • the genetically engineered bacteria comprise a gene encoding a propionate catabolism enzyme that is expressed under the control of an inducible promoter.
  • the genetically engineered bacterium that expresses a propionate catabolism enzyme and/or a transporter of propionate and /or metabolites thereof and/or propionate binding protein is under the control of a promoter that is activated by inflammatory conditions.
  • the gene for producing the propionate catabolism enzyme and/or a transporter of propionate and /or metabolites thereof and/or propionate binding protein 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.
  • 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 (•NO2), dinitrogen trioxide (N2O3), peroxynitrous acid
  • Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.
  • RNS-inducible regulatory region refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region.
  • the RNS-inducible regulatory region comprises a promoter sequence.
  • the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression.
  • 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 propionate catabolism enzyme gene sequence(s), e.g., any of the amino acid catabolism enzymes described herein.
  • a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence.
  • RNS induces expression of the gene or gene sequences.
  • 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.
  • 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., propionate catabolism enzyme gene sequence(s), propionate transporter sequence(s), propionate binding protein(s).
  • 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.
  • RNS derepresses expression of the gene or genes.
  • 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.
  • the RNS-repressible regulatory region comprises a promoter sequence.
  • the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence.
  • 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.
  • 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.
  • RNS represses expression of the gene or gene sequences.
  • a“RNS-responsive regulatory region” refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region.
  • 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.
  • 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 amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein, thus controlling expression of the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein relative to RNS levels.
  • the tunable regulatory region is a RNS-inducible regulatory region
  • the payload is an amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein, such as any of the amino acid catabolism enzymes, propionate transporters, and propionate binding proteins provided herein;
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene or genes.
  • inflammation is ameliorated, RNS levels are reduced, and production of the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is decreased or eliminated.
  • 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.
  • the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression.
  • 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.
  • the tunable regulatory region is a RNS-inducible regulatory region
  • the transcription factor that senses RNS is NorR.
  • 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;
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene sequence(s).
  • 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 amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • the tunable regulatory region is a RNS-inducible regulatory region
  • the transcription factor that senses RNS is DNR.
  • DNR dissimilatory nitrate respiration regulator
  • 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).
  • 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.
  • 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 amino acid catabolism enzymes.
  • the DNR is Pseudomonas aeruginosa DNR.
  • 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.
  • the tunable regulatory region is a RNS-derepressible regulatory region
  • the transcription factor that senses RNS is NsrR.
  • NsrR is“an Rrf2- type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism” (Isabella et al., 2009).
  • the genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is repressed by NsrR.
  • the NsrR is Neisseria gonorrhoeae NsrR.
  • the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a propionate catabolism enzyme gene or genes.
  • an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene or genes and producing the encoding an amino acid catabolism enzyme(s).
  • the genetically engineered bacteria 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.
  • 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.
  • the genetically engineered bacterium of the invention is Escherichia coli
  • 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.
  • the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
  • 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.
  • 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.
  • the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express an amino acid 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 an amino acid catabolism enzyme.
  • the RNS- sensing repressor inhibits transcription of the second repressor, which inhibits the
  • second repressors useful in these embodiments include, but are not limited to, TetR, C1, and LexA.
  • first repressor which occurs in the absence of RNS
  • the second repressor is transcribed, which represses expression of the gene or genes.
  • expression of the second repressor is repressed, and the gene or genes, e.g., a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene or genes is expressed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a RNS-sensing transcription factor e.g., the nsrR gene
  • an inducible promoter e.g., the GlnRS promoter or the P(Bla) promoter
  • expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule.
  • expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule.
  • the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.
  • 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.
  • the genetically engineered bacteria comprise a RNS- sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae.
  • NsrR RNS- sensing transcription factor
  • nsrR regulatory region from Neisseria gonorrhoeae.
  • the native RNS-sensing transcription factor e.g., NsrR
  • the native RNS-sensing transcription factor e.g., NsrR
  • 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.
  • the gene encoding the RNS-sensing transcription factor is present on a plasmid.
  • the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids.
  • 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.
  • 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.
  • 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 propionate catabolism enzyme in the presence of RNS, as compared to the wild-type regulatory region under the same conditions.
  • 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 propionate catabolism enzyme in the presence of RNS, as compared to the wild-type transcription factor under the same conditions.
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein in the presence of RNS.
  • 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.
  • 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.
  • any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites.
  • one or more copies of a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein 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 amino acid catabolism enzyme(s) and also permits fine-tuning of the level of expression.
  • 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 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.
  • the genetically engineered bacteria comprise a gene for producing a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein that is expressed under the control of an inducible promoter.
  • the genetically engineered bacterium that expresses a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein under the control of a promoter that is activated by conditions of cellular damage.
  • the gene for producing the propionate 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.
  • 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 (H2O2), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical (•OH), superoxide or superoxide anion (•O2-), singlet oxygen (1O2), ozone (O3), carbonate radical, peroxide or peroxyl radical (•O2-2), hypochlorous acid (HOCl), hypochlorite ion (OCl-), sodium hypochlorite (NaOCl), nitric oxide (NO•), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by•).
  • Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al., 2014).
  • 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.
  • the ROS-inducible regulatory region comprises a promoter sequence.
  • the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression.
  • 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 amino acid catabolism enzyme(s).
  • a transcription factor e.g., OxyR
  • ROS induces expression of the gene or genes.
  • 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.
  • 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 amino acid catabolism enzyme(s).
  • a transcription factor e.g., OhrR
  • ROS derepresses expression of the gene or gene cassette.
  • 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.
  • the ROS-repressible regulatory region comprises a promoter sequence.
  • the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence.
  • 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.
  • a transcription factor e.g., PerR
  • ROS represses expression of the gene or genes.
  • a“ROS-responsive regulatory region” refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region.
  • 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.
  • 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 an amino acid catabolism enzyme, thus controlling expression of the propionate catabolism enzyme relative to ROS levels.
  • the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is an amino acid 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 amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein thereby producing the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is decreased or eliminated.
  • 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.
  • the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression.
  • 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.
  • the tunable regulatory region is a ROS-inducible regulatory region
  • the transcription factor that senses ROS is OxyR.
  • 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 al., 2001; Dubbs et al., 2012; Table 1).
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene.
  • an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene and producing the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • OxyR is encoded by an E. coli oxyR gene.
  • the oxyS regulatory region is an E. coli oxyS regulatory region.
  • the ROS- inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.
  • the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR.
  • 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 H2O2.
  • the genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is activated by SoxR.
  • the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., an amino acid catabolism enzyme.
  • a gene e.g., an amino acid catabolism enzyme.
  • the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene and producing an amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • 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.
  • the tunable regulatory region is a ROS-derepressible regulatory region
  • the transcription factor that senses ROS is OhrR.
  • OhrR is a“transcriptional repressor [that]... senses both organic peroxides and NaOCl” (Dubbs et al., 2012) and is“weakly activated by H2O2 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).
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene.
  • a ROS e.g., NaOCl
  • an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene and producing the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • OhrR is a member of the MarR family of ROS-responsive regulators.“Most members of the MarR family are transcriptional repressors and often bind to the -10 or -35 region in the promoter causing a steric inhibition of RNA polymerase binding” (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ.
  • the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ
  • the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ.
  • Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012).
  • 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 H2O2” (Bussmann et al., 2010).
  • RosR is capable of repressing numerous genes and putative genes, including but not limited to“a putative polyisoprenoid-binding protein (cg1322, 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 (cg1426), two putative FMN reductases (cg1150 and cg1850), and four putative monooxygenases (cg0823, cg1848, cg2329, and cg3084)” (Bussmann et al., 2010).
  • a putative polyisoprenoid-binding protein cg1322, gene upstream of and divergent from rosR
  • cgtS9 a sensory histidine kinase
  • cgtS9 a putative transcriptional regulator of the Crp/
  • the genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010; Table 1).
  • 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 amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene and producing the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • the genetically engineered bacteria 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.
  • 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.
  • the genetically engineered bacterium of the invention is Escherichia coli
  • the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR.
  • the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
  • 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.
  • 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.
  • the tunable regulatory region is a ROS-repressible regulatory region
  • the transcription factor that senses ROS is PerR.
  • 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 H2O2” (Dubbs et al., 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 al., 2012).
  • the genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al., 2012; Table 1).
  • the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express an amino acid 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., an amino acid catabolism enzyme.
  • a first ROS-sensing repressor e.g., PerR
  • TetR e.g., TetR
  • the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette.
  • second repressors useful in these embodiments include, but are not limited to, TetR, C1, and LexA.
  • the ROS-sensing repressor is PerR.
  • the second repressor is TetR.
  • a PerR-repressible regulatory region drives expression of TetR
  • a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., an amino acid catabolism enzyme.
  • tetR 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., an amino acid 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., an amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed.
  • the gene or gene cassette e.g., an amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed.
  • a ROS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria.
  • 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 al., 2012)
  • the genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OxyR.
  • 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 al., 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.
  • the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by RosR.
  • the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by PerR.
  • ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria.
  • “OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012).
  • 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.
  • 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.
  • nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 6. OxyR binding sites are underlined and bolded.
  • genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 18, 19, 20, or 21, or a functional fragment thereof.
  • e gene ca y eng neere ac er a o e nven on 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.
  • 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.
  • 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.
  • 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.
  • the genetically engineered bacteria comprise a ROS- sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from
  • the native ROS-sensing transcription factor e.g., OxyR
  • the native ROS- sensing transcription factor e.g., OxyR
  • 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.
  • the gene encoding the ROS-sensing transcription factor is present on a plasmid.
  • the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids.
  • the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same.
  • the gene encoding the ROS- sensing transcription factor is present on a chromosome.
  • 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.
  • the genetically engineered bacteria comprise a wild- type gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein in the presence of ROS, as compared to the wild-type transcription factor under the same conditions.
  • 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 propionate catabolism enzyme in the presence of ROS.
  • the gene or gene cassette for producing the propionate 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 propionate 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 propionate catabolism enzyme is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.
  • the gene or gene cassette for producing the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.
  • 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.
  • the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing an amino acid catabolism enzyme(s), propionate transporter(s), and/or propionate binding protein(s).
  • the gene(s) capable of producing an amino acid catabolism enzyme(s), propionate transporter(s), and/or propionate binding protein(s) is present on a plasmid and operatively linked to a ROS- responsive regulatory region.
  • the gene(s) capable of producing a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is present in a chromosome and operatively linked to a ROS-responsive regulatory region.
  • the genetically engineered bacteria or genetically engineered virus produce one or more amino acid 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.
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing an amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein such that the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein 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.
  • a bacterium may comprise multiple copies of the gene encoding the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • the gene encoding the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed on a low-copy plasmid.
  • the low-copy plasmid may be useful for increasing stability of expression.
  • the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions.
  • the gene encoding the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing expression of the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.
  • the gene encoding the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed on a chromosome.
  • the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions.
  • MOAs mechanisms of action
  • the genetically engineered bacteria may include four copies of the gene encoding a particular propionate catabolism enzyme, propionate transporter, and/or propionate binding protein inserted at four different insertion sites.
  • the genetically engineered bacteria may include three copies of the gene encoding a particular propionate catabolism enzyme, propionate transporter, and/or propionate binding protein inserted at three different insertion sites and three copies of the gene encoding a different propionate catabolism enzyme, propionate transporter, and/or propionate binding protein inserted at three different insertion sites.
  • 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 amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.
  • qPCR quantitative PCR
  • Primers specific for propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art.
  • a fluorophore is added to a sample reaction mixture that may contain propionate catabolism enzymemRNA, 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene(s).
  • CT threshold cycle
  • qPCR quantitative PCR
  • Primers specific for propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art.
  • a fluorophore is added to a sample reaction mixture that may contain propionate catabolism enzyme, propionate transporter, and/or propionate binding protein 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
  • 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).
  • CT threshold cycle
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene(s).
  • the inducible promoter is a propionate responsive promoter.
  • the prpR promoter is a propionate responsive promoter.
  • the propionate responsive promoter comprises SEQ ID NO: 70.
  • the term“propionate catabolism gene,”“propionate catabolism gene cassette,”“propionate catabolism cassette”, or“propionate catabolism operon” refers to a gene or set of genes capable of catabolizing propionate, and/or a metabolite thereof, and/or methylmalonic acid, an/or a metabolite thereof, in a biosynthetic pathway.
  • the term“propionate catabolism enzyme” or“propionate catabolic or catabolism enzyme” or“propionate metabolic enzyme” refers to any enzyme that is capable of metabolizing propionate and/or a metabolite thereof.
  • the term“propionate catabolism enzyme” or“propionate catabolic or catabolism enzyme” or“propionate metabolic enzyme” refers to any enzyme that is capable of metabolizing methylmalonic acid and/or a metabolite thereof.
  • the term“propionate catabolism enzyme” or “propionate catabolic or catabolism enzyme” or“propionate metabolic enzyme” refers to any enzyme that is capable of metabolizing propionate, propionyl-CoA, methylmalonic acid, and/or methylmalonylCoA.
  • the term“propionate catabolism enzyme” or “propionate catabolic or catabolism enzyme” or“propionate metabolic enzyme” refers to any enzyme that is capable of reducing accumulated propionate and/or methylmalonic acid and/or propionylCoA and/or methylmalonylCoA or that can lessen, ameliorate, or prevent one or more propionate and/or methylmalonic acid diseases or disease symptoms.
  • propionate and/or methylmalonic acid metabolic enzymes include, but are not limited to, propionyl CoA carboxylase (PCC), methylmalonyl CoA mutase (MUT), propionyl-CoA synthetase (PrpE), 2-methylisocitrate lyase (PrpB), 2-methylcitrate synthase (prpC), 2- methylcitrate dehydratase (PrpD), propionyl-CoA carboxylase (pccB), Acetyl-/propionyl- coenzyme A carboxylase (accA1), Methylmalonyl-CoA epimerase (mmcE), methylmalonyl- CoA mutase (mutA, and mutB), Acetoacetyl-CoA reductase (phaB), Polyhydroxyalkanoic acid (PHA) synthases, e.g.,encoded by phaC, and 3-keto
  • Propionate catabolism enzymes of the present disclosure include both wild-type or modified propionate catabolism enzymes and can be produced using recombinant and synthetic methods or purified from nature sources. Propionate catabolism enzymes include full-length polypeptides and functional fragments thereof, as well as homologs and variants thereof.
  • Propionate catabolism enzymes include polypeptides that have been modified from the wild- type sequence, including, for example, polypeptides having one or more amino acid deletions, insertions, and/or substitutions and may include, for example, fusion polypeptides and polypeptides having additional sequence, e.g., regulatory peptide sequence, linker peptide sequence, and other peptide sequence.
  • the term“propionate catabolism enzyme” refers to an enzyme involved in the catabolism of propionate or propionyl CoA and or methylmalonic acid or methylmalonylCoA to a non-toxic molecule, such as its corresponding methylmalonyl CoA molecule, corresponding succinyl CoA molecule, succinate, or polyhydroxyalkanotes; or the catabolism of methylmalonyl CoA to non-toxic molecule, such as its corresponding succinyl CoA molecule.
  • Enzymes involved in the catabolism of propionate are well known to those of skill in the art.
  • propionyl CoA carboxylase PCC
  • MUT methylmalonyl CoA mutase
  • Enzyme deficiencies or mutations which lead to the toxic accumulation of propionyl CoA or methylmalonyl CoA result in the development of disorders associated with propionate catabolism, such as PA and MMA, and severe nutritional deficiencies of Vitamin B 12 can also result in MMA (Higginbottom et al., M. Engl. J.
  • the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionyl CoA carboxylase (PCC). In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionyl CoA carboxylase (PCC) and one or more copies of methylmalonyl CoA mutase (MUT).
  • PCC propionyl CoA carboxylase
  • MUT methylmalonyl CoA mutase
  • the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionyl-CoA synthetase (PrpE). In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionate CoA transferase (Pct).
  • the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionyl-CoA synthetase (PrpE) and one or more copies of propionyl CoA carboxylase (PCC). In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionyl-CoA synthetase (PrpE), one or more copies of propionyl CoA carboxylase (PCC) and one or more copies of methylmalonyl CoA mutase (MUT).
  • PrpE propionyl-CoA synthetase
  • PCC propionyl CoA carboxylase
  • the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionate CoA transferase (Pct) and one or more copies of propionyl CoA carboxylase (PCC). In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionate CoA transferase (Pct), one or more copies of propionyl CoA carboxylase (PCC) and one or more copies of methylmalonyl CoA mutase (MUT). [0226] PrpE converts propionate and free CoA to propionyl-CoA in an irreversible, ATP-dependent manner, releasing AMP and PPi (pyrophosphate).
  • PrpE can be inactivated by postranslational modification of the active site lysine, e.g., as shown in FIG.9A.
  • Protein lysine acetyltransferase (Pka) in E. coli carries out the propionylation of PrpE.
  • the enzyme CobB depropionylates PrpEPr making the inactivation reversible.
  • the inactivation pathway can be eliminated entirely through the deletion of the pka gene.
  • the genetically engineered bacteria comprise a deletion of pka ( ⁇ pka) to prevent the inactivation of PrpE.
  • the deletion of pka results in greater activity of PrpE and downstream catabolic enzymes.
  • Pct converts propionate and acetyl-CoA to propionyl-CoA and acetate in a reversible reaction.
  • the genetically engineered bacteria comprise a gene encoding Pct for the generation of propionylCoA from propionate, e.g., as shown in FIG.9B.
  • the genetically engineered bacteria comprise Pct in combination with or as a component of one or more of PHA and/or MMCA and/or 2MC pathway cassette(s).
  • PrpB, PrpC, and PrpD are capable of converting propionyl CoA into succinate and pyruvate
  • PrpB, PrpC, PrpD, and PrpE are capable of converting propionate into succinate and pyruvate.
  • PrpE a propionate-CoA ligase, converts propionate to propionyl CoA.
  • PrpC a 2-methylcitrate synthetase, then converts propionyl CoA to 2-methylcitrate.
  • the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpB, PrpC, and PrpD.
  • the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpB, PrpC, PrpD, and PrpE.
  • the engineered bacterium comprises two or more copies of a gene encoding any of the following: PrpB, PrpC, and PrpD, and combinations thereof.
  • the engineered bacterium comprises two or more copies of a gene encoding any of the following: PrpB, PrpC, PrpD, and PrpE, and combinations thereof.
  • the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpE, PhaA, and PhaB.
  • the disclosure encompasses the design of genetic circuits which mimic the functional activities of the human methylmalonyl-CoA pathway in order to catabolize propionate to treat diseases associated with propionate catabolism.
  • a circuit can be designed to express prpE, pccB, accA1, mmcE, mutA, and mutB (FIG.15).
  • PrpE converts propionate to propionyl-CoA, which is then converted to D-methylmalonyl-CoA by PccB and AccA1.
  • D-methylmalonyl-CoA is then converted to L-methylmalonyl-CoA by MmcE, and MutA and MutB convert L-methylmalonyl CoA to succinyl-CoA.
  • these genes can be split up into two circuits, i.e, prpE-accA1-pccB and mmcE-mutA-mutB, as indicated in FIG.15.
  • the engineered bacterium comprises gene sequence(s) selected from: prpE, pccB, accA1, mmcE, mutA, and mutB.
  • the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpE, PccB, AccA1, MmcE, MutA, and MutB.
  • the disclosure encompasses the design of genetic circuits which constitute the 2-methylcitrate cycle pathway in bacteria, such as the prpBCDE circuit (FIG.20) or the
  • polyhydroxyalkanoate pathway such as the prpE, phaB, phaC, phaA genes (FIG.10C) in order to catabolize propionate to treat diseases associated with propionate catabolism.
  • the disclosure encompasses the design of genetic circuits which comprise MatB.
  • Malonyl-coenzyme A (malonyl-CoA) synthetase (MatB) belongs to the AMP-forming acyl-CoA synthetase protein family.
  • MatB Synthetase of Rhodopseudomonas palustris; Appl. Environ. Microbiol. September 2012 vol.78 no.186619-6629, and references therein).
  • MatB converts malonate to malonyl- CoA in two steps according to this mechanism via a malonyl-AMP intermediate, and similarly also converts methylmalonate to methylmalonyl-CoA.
  • a genetic circuit comprising MatB is useful in the treatment of methylmalonic acidemia, allowing accumulated methylmalonic acid to be converted into
  • methylmalonylCoA Once converted to methylmalonylCoA, catabolism can proceed along the MMCA pathway (e.g., through mmcE, mutA, and mutB). Alternatively, methylmalonylCoA can be converted to propionylCoA. This reaction may be catalyzed by the AccA1/PccB complex, which is encoded by a genetic circuit of the disclosure. The AccA1/pccB complex catalyzes the reversible conversion of propionylCoA to
  • the engineered bacterium may further comprise gene sequence(s) encoding MatB.
  • one or more gene(s) or gene cassette(s) comprise MatB, e.g., MatB derived from Rhodopseudomonas palustris.
  • the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) comprising MatB, e.g., MatB derived from Rhodopseudomonas palustris.
  • genetically engineered bacteria comprising one or more gene(s) or gene cassettes comprising MatB are suitable for the treatment of methylmalonic acidemia or methylmalonic acidemia and propionic acidemia.
  • the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding MatB and one or more MMCA gene cassettes as described herein. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding MatB and one or more MMCA gene(s) or MMCA gene cassette(s) as described herein. In some embodiments, MatB is driven by a separate promoter and is on a separate plasmid or chromosomal integration site. In some
  • MatB part of an operon comprising one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes described herein.
  • the genetically engineered bacteria encode one or more of MatB, mmcE, mutA, and mutB. In some embodiments, the genetically engineered bacteria encode MatB, mmcE, mutA, and mutB. In some embodiments, a genetic circuit encoded by the genetically engineered bacteria comprises MatB, mmcE, mutA, and mutB.
  • the genetically engineered bacteria encode one or more of MatB, Acc1A, and PccB. In some embodiments, the genetically engineered bacteria encode MatB, Acc1A, and PccB. In some embodiments, a genetic circuit encoded by the genetically engineered bacteria comprises MatB, Acc1A, and PccB. In some embodiments, the genetically engineered bacteria encode MatB, Acc1A, and PccB, and mmcE, mutA and mutB. In some embodiments, the genetically engineered bacteria encode MatB, Acc1A, and PccB, and mmcE, mutA and mutB and further prpE.
  • the genetically engineered bacteria encode MatB, Acc1A, and PccB, and mmcE, mutA and mutB, and further encode a PHA and/or 2MC pathway circuit, and may or may not further comprise prpE.
  • These genes may be organized in one or more gen cassettes, as described herein.
  • Non- limiting examples of genetically engineered bacteria comprising one or more gene(s) or gene cassettes and comprising exemplary operons or gene cassette(s) are depicted in FIG.21G and FIG.21F.
  • the one or more gene cassettes may be organized as follows; MatB-mmcE-mutA-mutB; MatB-Acc1A-PccB and mmcE-mutA-mutB, alone or in combination with PPHA and/or 2MC pathway cassettes; PrpE-MatB-Acc1A-PccB and mmcE-mutA-mutB, alone or in combination with PPHA and/or 2MC pathway cassettes.
  • expression of the propionate catabolism gene cassette increases the rate of propionate, propionyl CoA, and/or methylmalonyl CoA catabolism in the cell. In one embodiment, expression of the propionate catabolism gene cassette decreases the level of propionate in the cell. In another embodiment, expression of the propionate catabolism gene cassette decreases the level of propionic acid in the cell. In one
  • expression of the propionate catabolism gene cassette decreases the level of propionyl CoA in the cell. In one embodiment, expression of the propionate catabolism gene cassette decreases the level of methylmalonyl CoA in the cell. In one embodiment, expression of the propionate catabolism gene cassette decreases the level of methylmalonic acid in the cell.
  • expression of the propionate catabolism gene cassette increases the level of methylmalonyl CoA in the cell as compared to the level of its corresponding propionyl CoA in the cell.
  • expression of the propionate catabolism gene cassette increases the level of succinate in the cell as compared to the level of its corresponding methylmalonyl CoA in the cell.
  • expression of the propionate catabolism gene cassette decreases the level of the propionate, propionyl CoA, and/or methylmalonyl CoA as compared to the level of succinate or succinyl CoA in the cell.
  • expression of the propionate catabolism gene cassette increases the level of succinate or succinyl CoA in the cell as compared to the level of the propionate, propionyl CoA, and/or methylmalonyl CoA in the cell.
  • Enzymes involved in the catabolism of propionate may be expressed or modified in the bacteria in order to enhance catabolism of propionate.
  • the heterologous propionate catabolism gene or gene cassette when expressed in the engineered bacterial cells, the bacterial cells convert more propionate and/or propionyl CoA into methylmalonyl CoA, or convert more methylmalonyl CoA into succinate or succinyl CoA when the gene or gene cassette is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria expressing a heterologous propionate catabolism gene or gene cassette can catabolize propionate, propionyl CoA, and/or methylmalonyl CoA to treat diseases associated with catabolism of propionate, such as Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA).
  • PA Propionic Acidemia
  • MMA Methylmalonic Acidemia
  • the expression of the propionate catabolism gene cassette decreases the levels of one or more propionic acidemia and/or methylmalonic acidemia biomarkers.
  • the propionate catabolism gene cassette expressed by the genetically engineered bacteria decreases the levels of one or more propionic acidemia and/or methylmalonic acidemia biomarkers.
  • expression of the propionate catabolism gene cassette decreases the propionylcarnitine to acetylcarnitine ratio in the blood and/or the urine, e.g., in a mammalian subject with elevated levels of propionate and/or methylmalonate.
  • expression of the propionate catabolism gene cassette decreases levels of 2-methylcitrate in the blood and/or in the urine, e.g., in a mammalian subject with elevated levels of propionate and/or
  • expression of the propionate catabolism gene cassette decreases levels of propionylglycine in the blood and/or in the urine, e.g., in a mammalian subject with elevated levels of propionate and/or methylmalonate. In one embodiment, expression of the propionate catabolism gene cassette decreases levels of tiglyglycine in the blood and/or in the urine, e.g., in a mammalian subject with elevated levels of propionate and/or methylmalonate.
  • the bacterial cell comprises at least one heterologous gene encoding at least one propionate catabolism enzyme. In one embodiment, the bacterial cell comprises at least one heterologous gene encoding a transporter of propionate and at least one heterologous gene encoding at least one propionate catabolism enzyme.
  • the engineered bacterial cell comprises at least one heterologous gene or gene cassette encoding at least one propionate catabolism enzyme.
  • the disclosure provides a bacterial cell that comprises at least one heterologous gene or gene cassette encoding at least one propionate catabolism enzyme operably linked to a first promoter.
  • the bacterial cell comprises at least one gene or gene cassette encoding at least one propionate catabolism enzyme from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises more than one copy of a native gene or gene cassette encoding one or more propionate catabolism enzyme(s).
  • the bacterial cell comprises at least one native gene or gene cassette encoding at least one native propionate catabolism enzyme, as well as at least one copy of at least one gene or gene cassette encoding one or more propionate catabolism enzyme(s) from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene or gene cassette encoding one or more propionate catabolism enzyme(s).
  • the bacterial cell comprises multiple copies of a gene or gene cassette encoding one or more propionate catabolism enzyme(s).
  • a gene cassette may comprise one or more native and one or more non-native or heterologous genes.
  • propionate catabolism enzyme is encoded by at least one gene encoding at least one propionate catabolism enzyme derived from a bacterial species.
  • a propionate catabolism enzyme is encoded by one or more gene(s) or gene cassettes encoding a propionate catabolism enzyme derived from a non-bacterial species.
  • a propionate catabolism enzyme is encoded by a gene derived from a eukaryotic species, e.g., a yeast species or a plant species. In one
  • a propionate catabolism enzyme is encoded by a gene derived from a human.
  • the at least one gene encoding the at least one propionate catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to, Acetinobacter, Azospirillum, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Burkholderia, Citrobacter, Clostridium, Corynebacterium, Cronobacter, Enterobacter, Enterococcus, Erwinia, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Leishmania, Listeria, Macrococcus, Mycobacterium, Nakamurella, Nasonia, Nostoc, Pantoea,
  • Pectobacterium Pseudomonas, Psychrobacter, Ralstonia, Saccharomyces, Salmonella, Sarcina, Serratia, Staphylococcus, and Yersinia, e.g., Acetinobacter radioresistens,
  • Acetinobacter baumannii Acetinobacter calcoaceticus, Azospirillum brasilense, Bacillus anthracis, Bacillus cereus, Bacillus coagulans, Bacillus megaterium,Bacillus subtilis, Bacillus thuringiensis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Burkholderia xenovorans, Citrobacter youngae, Citrobacter koseri, Citrobacter rodentium, Clostridium acetobutylicum, Clostridium butyricum,
  • Corynebacterium aurimucosum Corynebacterium kroppenstedtii, Corynebacterium striatum, Cronobacter sakazakii, Cronobacter turicensis, Enterobacter cloacae, Enterobacter cancerogenus, Enterococcus faecium, Erwinia amylovara, Erwinia pyrifoliae, Erwinia tasmaniensis, Helicobacter mustelae, Klebsiella pneumonia, Klebsiella variicola,
  • Lactobacillus acidophilus Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri,
  • Lactobacillus rhamnosus Lactococcus lactis, Leishmania infantum, Leishmania major, Leishmania brazilensis, Listeria grayi, Macrococcus caseolyticus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium leprae,
  • Mycobacterium marinum Mycobacterium smegmatis, Mycobacterium tuberculosis,
  • Mycobacterium ulcerans Nakamurella multipartita, Nasonia vitipennis, Nostoc punctiforme, Pantoea ananatis, Pantoea agglomerans, Pectobacterium atrosepticum, Pectobacterium carotovorum, Pseudomonas aeruginosa, Psychrobacter articus, Psychrobacter
  • cryohalolentis Ralstonia eutropha, Saccharomyces boulardii, Salmonella enterica, Sarcina ventriculi, Serratia odorifera, Serratia proteamaculans, Staphylococcus aerus,
  • Staphylococcus capitis Staphylococcys carnosus, Staphylococcus epidermidis,
  • Staphylococcus hominis Staphylococcus haemolyticus, Staphylococcus lugdunensis,
  • Staphylococcus saprophyticus Staphylococcus warneri, Yersinia enterocolitica, Yersinia mollaretii, Yersinia kristensenii, Yersinia rohdei, and Yersinia aldovae.
  • the gene encoding prpE is derived from E. coli.
  • the gene encoding accA1 is derived from Streptopmyces coelicolor.
  • the gene encoding pccB is derived from E. coli.
  • the gene encoding mmcE is derived from Propionibcterium freudenheimii.
  • the gene encoding mutA is derived from Propionibcterium freudenheimii.
  • the gene encoding mutB is derived from Propionibcterium
  • the gene encoding prpB is derived from E. coli. In some embodiments, the gene encoding prpC is derived from E. coli. In some embodiments, the gene encoding prpD is derived from E. coli. In some embodiments, the gene encoding phaB is derived from Acinetobacter sp RA3849. In some embodiments, the gene encoding phaC is derived from Acinetobacter sp RA3849. In some embodiments, the gene encoding phaA is derived from Acinetobacter sp RA3849.
  • the at least one gene encoding the at least one propionate catabolism enzyme has been codon-optimized for use in the engineered bacterial cell.
  • the at least one gene or gene cassette encoding the one or more propionate catabolism enzyme(s) has been codon-optimized for use in Escherichia coli.
  • the at least one gene encoding the at least one propionate catabolism enzyme is expressed in the engineered bacterial cells, the bacterial cells catabolize more propionate or propionyl CoA than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions).
  • the genetically engineered bacteria comprising at least one heterologous gene or gene cassette encoding one or more propionate catabolism enzyme(s) may be used to catabolize excess propionate, propionic acid, and/or propionyl CoA to treat a disease associated with the catabolism of propionate, such as Propionic Acidemia, Methylmalonic Acidemia, or a vitamin B 12 deficiency.
  • a disease associated with the catabolism of propionate such as Propionic Acidemia, Methylmalonic Acidemia, or a vitamin B 12 deficiency.
  • the present disclosure further comprises genes and gene cassettes encoding functional fragments of a propionate catabolism enzyme or functional variants of a propionate catabolism enzyme(s).
  • the term“functional fragment thereof” or “functional variant thereof” of a propionate catabolism enzyme relates to an element having qualitative biological activity in common with the wild-type propionate catabolism enzyme from which the fragment or variant was derived.
  • a functional fragment or a functional variant of a mutated propionate catabolism enzyme is one which retains essentially the same ability to catabolize propionyl CoA and/or methylmalonyl CoA as the propionate catabolism enzyme from which the functional fragment or functional variant was derived.
  • a polypeptide having propionate catabolism enzyme activity may be truncated at the N-terminus or C-terminus and the retention of propionate catabolism enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein.
  • the engineered bacterial cell comprises a heterologous gene encoding a propionate catabolism enzyme functional variant.
  • the engineered bacterial cell comprises a heterologous gene or gene cassette encoding a propionate catabolism enzyme functional fragment.
  • Assays for testing the activity of a propionate catabolism enzyme, a propionate catabolism enzyme functional variant, or a propionate catabolism enzyme functional fragment are well known to one of ordinary skill in the art.
  • propionate catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in an engineered bacterial cell that lacks endogenous propionate catabolism enzyme activity.
  • propionate can be supplemented in the media, and engineered bacterial strains can be compared with corresponding wild type strains with respect to propionate depletion from the media, as described herein.
  • Propionate levels can be assessed using mass spectrometry or gas chromatography.
  • samples can be injected into a Perkin Elmer Autosystem XL Gas Chromatograph containing a Supelco packed column, and the analysis can be performed according to manufacturing instructions (see, for example, Supelco I (1998) Analyzing fatty acids by packed column gas chromatography, Bulletin 856B:2014).
  • propionate levels can be determined using high-pressure liquid
  • HPLC chromatography
  • a computer-controlled Waters HPLC system equipped with a model 600 quaternary solvent delivery system, and a model 996 photodiode array detector, and components of a sample can be resolved with an Aminex HPX-87H (300 by 7.8 mm) organic acid analysis column (Bio-Rad Laboratories) (see, for example, Palacios et al., 2003, J. Bacteriol., 185(9):2802-2810).
  • levels of certain propionate byproducts or metabolites e.g., propionylcarnitine/acetylcarnitine ratios, 2-methyl-citrate, propionylglycine, and/or tiglyglycine, can be measured in addition to propionate levels by mass spec as described 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.
  • the present disclosure encompasses genes encoding a propionate 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.
  • replacing a basic amino acid with another basic amino acid e.g., replacement among Lys, Arg, His
  • 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, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).
  • the gene(s) or gene cassette(s) encoding propionate catabolism enzyme(s) are mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene(s) or mutagenized gene cassettes) encoding the propionate catabolism enzyme(s) are isolated and inserted into the bacterial cell.
  • spontaneous mutants that arise that allow bacteria to grow on propionate as the sole carbon source can be screened for and selected.
  • the gene(s) comprising the modifications described herein may be present on a plasmid or chromosome.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpE.
  • prpE encodes PrpE, a propionate-CoA ligase. Accordingly, in one embodiment, the prpE gene has at least about 80% identity with SEQ ID NO: 25. In another embodiment, the prpE gene has at least about 80% identity with SEQ ID NO: 73. Accordingly, in one embodiment, the prpE gene has at least about 90% identity with SEQ ID NO: 25. In another embodiment, the prpE gene has at least about 90% identity with SEQ ID NO: 73. Accordingly, in one embodiment, the prpE gene has at least about 95% identity with SEQ ID NO: 25.
  • the prpE gene has at least about 95% identity with SEQ ID NO: 73. Accordingly, in one embodiment, the prpE gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. In another embodiment, the prpE gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 73. In another embodiment, the prpE gene comprises the sequence of SEQ ID NO: 25.
  • the prpE gene comprises the sequence of SEQ ID NO: 73. In yet another embodiment the prpE gene consists of the sequence of SEQ ID NO: 25. In another embodiment, the prpE gene consists of the sequence of SEQ ID NO: 73.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpC.
  • prpC encodes PrpC, a 2-methylcitrate synthetase. Accordingly, in one embodiment, the prpC gene has at least about 80% identity with SEQ ID NO: 57. In another embodiment, the prpC gene has at least about 80% identity with SEQ ID NO:76. Accordingly, in one embodiment, the prpC gene has at least about 90% identity with SEQ ID NO: 57. In another embodiment, the prpC gene has at least about 90% identity with SEQ ID NO: 76. Accordingly, in one embodiment, the prpC gene has at least about 95% identity with SEQ ID NO: 57.
  • the prpC gene has at least about 95% identity with SEQ ID NO: 76. Accordingly, in one embodiment, the prpC gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. In another embodiment, the prpC gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 76. In another embodiment, the prpC gene comprises the sequence of SEQ ID NO: 57.
  • the prpC gene comprises the sequence of SEQ ID NO: 76. In yet another embodiment the prpC gene consists of the sequence of SEQ ID NO: 57. In another embodiment, the prpC gene consists of the sequence of SEQ ID NO: 76.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpD.
  • prpD encodes PrpD, a 2-methylcitrate dehydrogenase.
  • the prpD gene has at least about 80% identity with SEQ ID NO: 58. In another embodiment, the prpD gene has at least about 80% identity with SEQ ID NO: 79. Accordingly, in one embodiment, the prpD gene has at least about 90% identity with SEQ ID NO: 58. In another embodiment, the prpD gene has at least about 90% identity with SEQ ID NO: 79. Accordingly, in one embodiment, the prpD gene has at least about 95% identity with SEQ ID NO: 58. In another embodiment, the prpD gene has at least about 95% identity with SEQ ID NO: 79.
  • the prpD gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. In another embodiment, the prpD gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 79. In another embodiment, the prpD gene comprises the sequence of SEQ ID NO: 58. In another embodiment, the prpD gene comprises the sequence of SEQ ID NO: 79. In yet another embodiment the prpD gene consists of the sequence of SEQ ID NO: 58. In another embodiment, the prpD gene consists of the sequence of SEQ ID NO: 79.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpB.
  • prpB encodes PrpB, a 2-methylisocitrate lyase. Accordingly, in one embodiment, the prpB gene has at least about 80% identity with SEQ ID NO: 56. In another embodiment, the prpB gene has at least about 80% identity with SEQ ID NO: 82. Accordingly, in one embodiment, the prpB gene has at least about 90% identity with SEQ ID NO: 56. In another embodiment, the prpB gene has at least about 90% identity with SEQ ID NO: 82. Accordingly, in one embodiment, the prpB gene has at least about 95% identity with SEQ ID NO: 56.
  • the prpB gene has at least about 95% identity with SEQ ID NO: 82. Accordingly, in one embodiment, the prpB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. In another embodiment, the prpB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 82. In another embodiment, the prpB gene comprises the sequence of SEQ ID NO: 56.
  • the prpB gene comprises the sequence of SEQ ID NO: 82. In yet another embodiment the prpB gene consists of the sequence of SEQ ID NO: 56. In another embodiment, the prpB gene consists of the sequence of SEQ ID NO: 82.
  • the at least one gene encoding the at least one propionate catabolism enzyme is phaB.
  • phaB encodes PhaB, a acetoacetyl-CoA reductase.
  • the phaB gene has at least about 80% identity with SEQ ID NO: 26. In one embodiment, the phaB gene has at least about 90% identity with SEQ ID NO: 26. In another embodiment, the phaB gene has at least about 95% identity with SEQ ID NO: 26. Accordingly, in one embodiment, the phaB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26. In another embodiment, the phaB gene comprises SEQ ID NO: 26. In yet another embodiment the phaB gene consists of SEQ ID NO: 26. [0256] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is phaC. phaC encodes PhaC, a polyhydroxyalkanoate synthase.
  • the phaC gene has at least about 80% identity SEQ ID NO: 27. In one embodiment, the phaC gene has at least about 90% identity with SEQ ID NO: 27. In another embodiment, the phaC gene has at least about 95% identity with SEQ ID NO: 27. Accordingly, in one embodiment, the phaC gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27. In another embodiment, the phaC gene comprises SEQ ID NO: 27. In yet another embodiment the phaC gene consists of SEQ ID NO: 27.
  • the at least one gene encoding the at least one propionate catabolism enzyme is phaA.
  • phaA encodes PhaA, a beta-ketothiolase. Accordingly, in one embodiment, the phaA gene has at least about 80% identity with a sequence which encodes SEQ ID NO: 28. In one embodiment, the phaA gene has at least about 90% identity with a sequence which encodes SEQ ID NO: 28. In another embodiment, the phaA gene has at least about 95% identity with a sequence which encodes SEQ ID NO: 28.
  • the phaA gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a sequence which encodes SEQ ID NO: 28.
  • the phaA gene comprises a sequence which encodes SEQ ID NO: 28.
  • the phaA gene consists of a sequence which encodes SEQ ID NO: 28.
  • the at least one gene encoding the at least one propionate catabolism enzyme is pccB.
  • pccB encodes PccB, a propionyl CoA carboxylase.
  • the pccB gene has at least about 80% identity with SEQ ID NO: 39. In one embodiment, the pccB gene has at least about 90% identity with SEQ ID NO: 39. In one embodiment, the pccB gene has at least about 95% identity with SEQ ID NO: 39. In one embodiment, the pccB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. In another embodiment, the pccB gene comprises the sequence of SEQ ID NO: 39. In yet another embodiment, the pccB gene consists of the sequence of SEQ ID NO: 39.
  • the at least one gene encoding the at least one propionate catabolism enzyme is pccB. Accordingly, in one embodiment, the pccB gene has at least about 80% identity with SEQ ID NO: 96. In one embodiment, the pccB gene has at least about 90% identity with SEQ ID NO: 96. In one embodiment, the pccB gene has at least about 95% identity with SEQ ID NO: 96. In one embodiment, the pccB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96. In another embodiment, the pccB gene comprises the sequence of SEQ ID NO: 96. In yet another embodiment, the pccB gene consists of the sequence of SEQ ID NO: 96.
  • the at least one gene encoding the at least one propionate catabolism enzyme is accA1.
  • accA1 encodes AccA1, an acetyl CoA carboxylase.
  • the accA1 gene has at least about 80% identity with SEQ ID NO: 38. In one embodiment, the accA1 gene has at least about 90% identity with SEQ ID NO: 38. In one embodiment, the accA1 gene has at least about 95% identity with SEQ ID NO: 38. In one embodiment, the accA1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38. In another embodiment, the accA1 gene comprises the sequence of SEQ ID NO: 38. In yet another embodiment, the accA1 gene consists of the sequence of SEQ ID NO: 38.
  • the at least one gene encoding the at least one propionate catabolism enzyme is accA1.
  • accA1 encodes AccA1, an acetyl CoA carboxylase.
  • the accA1 gene has at least about 80% identity with SEQ ID NO: 104. In one embodiment, the accA1 gene has at least about 90% identity with SEQ ID NO: 104. In one embodiment, the accA1 gene has at least about 95% identity with SEQ ID NO: 104. In one embodiment, the accA1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 104. In another embodiment, the accA1 gene comprises the sequence of SEQ ID NO: 104. In yet another embodiment, the accA1 gene consists of the sequence of SEQ ID NO: 104.
  • the at least one gene encoding the at least one propionate catabolism enzyme is mmcE.
  • mmcE encodes MmcE, a methylmalonyl-CoA mutase.
  • the mmcE gene has at least about 80% identity with SEQ ID NO: 32. In one embodiment, the mmcE gene has at least about 90% identity with SEQ ID NO: 32. In one embodiment, the mmcE gene has at least about 95% identity with SEQ ID NO: 32. In one embodiment, the mmcE gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 32. In another embodiment, the mmcE gene comprises the sequence of SEQ ID NO: 32. In yet another embodiment, the mmcE gene consists of the sequence of SEQ ID NO: 32.
  • the at least one gene encoding the at least one propionate catabolism enzyme is mmcE. Accordingly, in one embodiment, the mmcE gene has at least about 80% identity with SEQ ID NO: 106. In one embodiment, the mmcE gene has at least about 90% identity with SEQ ID NO: 106. In one embodiment, the mmcE gene has at least about 95% identity with SEQ ID NO: 106. In one embodiment, the mmcE gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 106. In another embodiment, the mmcE gene comprises the sequence of SEQ ID NO: 106. In yet another embodiment, the mmcE gene consists of the sequence of SEQ ID NO: 106.
  • the at least one gene encoding the at least one propionate catabolism enzyme is mutA.
  • mutA encodes MutA, a methylmalonyl-CoA mutase small subunit. Accordingly, in one embodiment, the mutA gene has at least about 80% identity with SEQ ID NO: 33. In one embodiment, the mutA gene has at least about 90% identity with SEQ ID NO: 33. In one embodiment, the mutA gene has at least about 95% identity with SEQ ID NO: 33.
  • the mutA gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 33.
  • the mutA gene comprises the sequence of SEQ ID NO: 33.
  • the mutA gene consists of the sequence of SEQ ID NO: 33.
  • the at least one gene encoding the at least one propionate catabolism enzyme is mutA. Accordingly, in one embodiment, the mutA gene has at least about 80% identity with SEQ ID NO: 110. In one embodiment, the mutA gene has at least about 90% identity with SEQ ID NO: 110. In one embodiment, the mutA gene has at least about 95% identity with SEQ ID NO: 110. In one embodiment, the mutA gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 110. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO: 110. In yet another embodiment, the mutA gene consists of the sequence of SEQ ID NO: 110.
  • the at least one gene encoding the at least one propionate catabolism enzyme is mutB.
  • mutB encodes MutB, a methylmalonyl-CoA mutase large subunit. Accordingly, in one embodiment, the mutB gene has at least about 80% identity with SEQ ID NO: 34. In one embodiment, the mutB gene has at least about 90% identity with SEQ ID NO: 34. In one embodiment, the mutB gene has at least about 95% identity with SEQ ID NO: 34.
  • the mutB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 34.
  • the mutB gene comprises the sequence of SEQ ID NO: 34.
  • the mutB gene consists of the sequence of SEQ ID NO: 34.
  • the at least one gene encoding the at least one propionate catabolism enzyme is mutB.
  • mutB encodes MutB, a methylmalonyl-CoA mutase large subunit. Accordingly, in one embodiment, the mutB gene has at least about 80% identity with SEQ ID NO: 112. In one embodiment, the mutB gene has at least about 90% identity with SEQ ID NO: 112. In one embodiment, the mutB gene has at least about 95% identity with SEQ ID NO: 112.
  • the mutB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 112.
  • the mutB gene comprises the sequence of SEQ ID NO: 112.
  • the mutB gene consists of the sequence of SEQ ID NO: 112.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpE. In one embodiment, the at least one propionate catabolism enzyme is prpE. In one embodiment, prpE has at least about 80% identity with SEQ ID NO: 71. In one embodiment, prpE has at least about 90% identity with SEQ ID NO: 71. In another embodiment, prpE has at least about 95% identity with SEQ ID NO: 71.
  • the prpE has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 71.
  • the prpE comprises a sequence which encodes SEQ ID NO: 71.
  • prpE consists of a sequence which encodes SEQ ID NO: 71.
  • the at least one propionate catabolism enzyme is phaA.
  • phaB has at least about 80% identity with SEQ ID NO: 137.
  • phaA has at least about 90% identity with SEQ ID NO: 175.
  • phaA has at least about 95% identity with SEQ ID NO: 137.
  • phaA has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 137.
  • phaA comprises a sequence which encodes SEQ ID NO: 137.
  • phaA consists of a sequence which encodes SEQ ID NO: 137.
  • the at least one propionate catabolism enzyme is phaB. Accordingly, in one embodiment, phaB has at least about 80% identity with SEQ ID NO: 135. In one embodiment, phaB has at least about 90% identity with SEQ ID NO: 135. In another embodiment, phaB has at least about 95% identity with SEQ ID NO: 135.
  • phaB has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 135.
  • phaB comprises a sequence which encodes SEQ ID NO: 135.
  • phaB consists of a sequence which encodes SEQ ID NO: 135.
  • the at least one propionate catabolism enzyme is phaC. Accordingly, in one embodiment, phaC has at least about 80% identity with SEQ ID NO: 136. In one embodiment, phaC has at least about 90% identity with SEQ ID NO: 136. In another embodiment, phaC has at least about 95% identity with SEQ ID NO: 136.
  • phaC has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 136.
  • phaC comprises a sequence which encodes SEQ ID NO: 136.
  • phaC consists of a sequence which encodes SEQ ID NO: 136.
  • the at least one propionate catabolism enzyme is mmcE. Accordingly, in one embodiment, mmcE has at least about 80% identity with SEQ ID NO: 132. In one embodiment, mmcE has at least about 90% identity with SEQ ID NO: 132. In another embodiment, mmcE has at least about 95% identity with SEQ ID NO: 132.
  • mmcE has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 132.
  • mmcE comprises a sequence which encodes SEQ ID NO: 132.
  • mmcE consists of a sequence which encodes SEQ ID NO: 132.
  • the at least one propionate catabolism enzyme is mutA. Accordingly, in one embodiment, mutA has at least about 80% identity with SEQ ID NO: 133. In one embodiment, mutA has at least about 90% identity with SEQ ID NO: 133. In another embodiment, mutA has at least about 95% identity with SEQ ID NO: 133.
  • mutA has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 133.
  • mutA comprises a sequence which encodes SEQ ID NO: 133.
  • mutA consists of a sequence which encodes SEQ ID NO: 133.
  • the at least one propionate catabolism enzyme is mutB. Accordingly, in one embodiment, mutB has at least about 80% identity with SEQ ID NO: 134. In one embodiment, mutB has at least about 90% identity with SEQ ID NO: 134. In another embodiment, mutB has at least about 95% identity with SEQ ID NO: 134. Accordingly, in one embodiment, mutB has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 134. In another embodiment, mutB comprises a sequence which encodes SEQ ID NO: 134. In yet another embodiment mutB consists of a sequence which encodes SEQ ID NO: 134.
  • the at least one propionate catabolism enzyme is accA. Accordingly, in one embodiment, accA has at least about 80% identity with SEQ ID NO: 130. In one embodiment, accA has at least about 90% identity with SEQ ID NO: 130. In another embodiment, accA has at least about 95% identity with SEQ ID NO: 130.
  • accA has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 130.
  • accA comprises a sequence which encodes SEQ ID NO: 130.
  • the accA consists of a sequence which encodes SEQ ID NO: 130.
  • the at least one propionate catabolism enzyme is pccB. Accordingly, in one embodiment, pccB has at least about 80% identity with SEQ ID NO: 131. In one embodiment, pccB has at least about 90% identity with SEQ ID NO: 131. In another embodiment, pccB has at least about 95% identity with SEQ ID NO: 131.
  • pccB has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 131.
  • pccB comprises a sequence which encodes SEQ ID NO: 131.
  • pccB consists of a sequence which encodes SEQ ID NO: 131.
  • the at least one propionate catabolism enzyme is prpC.
  • prpC has at least about 80% identity with SEQ ID NO: 74.
  • prpC has at least about 90% identity with SEQ ID NO: 74.
  • prpC has at least about 95% identity with SEQ ID NO: 74.
  • prpC has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 74.
  • prpC comprises a sequence which encodes SEQ ID NO: 74.
  • prpC consists of a sequence which encodes SEQ ID NO: 74.
  • the at least one propionate catabolism enzyme is prpD.
  • prpD has at least about 80% identity with SEQ ID NO: 77.
  • prpD has at least about 90% identity with SEQ ID NO: 77.
  • prpD has at least about 95% identity with SEQ ID NO: 77.
  • prpD has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 77.
  • prpD comprises a sequence which encodes SEQ ID NO: 77.
  • prpD consists of a sequence which encodes SEQ ID NO: 77.
  • the at least one gene encoding the at least one propionate catabolism enzyme is MatB.
  • MatB encodes Malonyl-coenzyme A (malonyl-CoA) synthetase (MatB).
  • the MatB gene has at least about 80% identity with SEQ ID NO: 141.
  • the MatB gene has at least about 90% identity with SEQ ID NO: 141.
  • the MatB gene has at least about 95% identity with SEQ ID NO: 141.
  • the MatB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 141.
  • the MatB gene comprises the sequence of SEQ ID NO: 141.
  • the MatB gene consists of the sequence of SEQ ID NO: 141.
  • the at least one propionate catabolism enzyme is matB. Accordingly, in one embodiment, matB has at least about 89% identity with SEQ ID NO: 140. In one embodiment, matB has at least about 90% identity with SEQ ID NO: 140. In another embodiment, matB has at least about 95% identity with SEQ ID NO: 140.
  • matB has at least about 85%, 86%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 140.
  • matB comprises a sequence which encodes SEQ ID NO: 140.
  • matB consists of a sequence which encodes SEQ ID NO: 140.
  • the at least one propionate catabolism enzyme is prpB.
  • prpB has at least about 80% identity with SEQ ID NO: 80.
  • prpB has at least about 90% identity with SEQ ID NO: 80.
  • prpB has at least about 95% identity with SEQ ID NO: 80.
  • prpB has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 80.
  • prpB comprises a sequence which encodes SEQ ID NO: 80.
  • prpB consists of a sequence which encodes SEQ ID NO: 80.
  • any combination of propionate catabolism enzymes that effectively reduce the level of propionate and/or a metabolite thereof can be used.
  • any combination of propionate catabolism enzymes that effectively reduce levels of propionate, propionyl CoA, and/or methylmalonyl CoA in a subject can be used.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpBCD.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpBCDE.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpE, pccB, accA1, mmcE, mutA, and mutB.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpE, pccB, and accA1 under the control of a first inducible promoter, and mmcE, mutA, and mutB under the control of a second inducible promoter.
  • the at least one gene encoding the at least one propionate catabolism enzyme is prpE, phaB, phaC, and phaA.
  • the propionate catabolism gene cassette comprises prpBCD. Accordingly, in one embodiment, the prpBCD operon has at least about 80% identity with SEQ ID NO: 138. In another embodiment, the prpBCD operon has at least about 80% identity with SEQ ID NO: 83 OR SEQ ID NO: 84. Accordingly, in one embodiment, the prpBCD operon has at least about 90% identity with SEQ ID NO: 138. In another embodiment, the prpBCD operon has at least about 90% identity with SEQ ID NO: 83 OR SEQ ID NO: 84. Accordingly, in one embodiment, the prpBCD operon has at least about 95% identity with SEQ ID NO: 138.
  • the prpBCD operon has at least about 95% identity with SEQ ID NO: 83 OR SEQ ID NO: 84. Accordingly, in one embodiment, the prpBCD operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 138. In another embodiment, the prpBCD operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 83 OR SEQ ID NO: 84.
  • the prpBCD operon comprises the sequence of SEQ ID NO: 138. In another embodiment, the prpBCD operon comprises the sequence of SEQ ID NO: 83 OR SEQ ID NO: 84. In yet another embodiment the prpBCD operon consists of the sequence of SEQ ID NO: 138. In another embodiment, the prpBCD operon consists of the sequence of SEQ ID NO: 83 OR SEQ ID NO: 84.
  • the propionate catabolism gene cassette comprises prpBCDE. Accordingly, in one embodiment, the prpBCDE operon has at least about 80% identity with SEQ ID NO: 55. In another embodiment, the prpBCDE operon has at least about 80% identity with SEQ ID NO: 93 or SEQ ID NO: 94. Accordingly, in one embodiment, the prpBCDE operon has at least about 90% identity with SEQ ID NO: 55. In another embodiment, the prpBCDE operon has at least about 90% identity with SEQ ID NO: 93 or SEQ ID NO: 94. Accordingly, in one embodiment, the prpBCDE operon has at least about 95% identity with SEQ ID NO: 55.
  • the prpBCDE operon has at least about 95% identity with SEQ ID NO: 93 or SEQ ID NO: 94. Accordingly, in one embodiment, the prpBCDE operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. In another embodiment, the prpBCDE operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 93 or SEQ ID NO: 94.
  • the prpBCDE operon comprises the sequence of SEQ ID NO: 55. In another embodiment, the prpBCDE operon comprises the sequence of SEQ ID NO: 93 or SEQ ID NO: 94. In yet another embodiment the prpBCDE operon consists of the sequence of SEQ ID NO: 55. In another embodiment, the prpBCDE operon consists of the sequence of SEQ ID NO: 93 or SEQ ID NO: 94.
  • the propionate catabolism gene cassette comprises phaBCA.
  • the phaBCA operon has at least about 80% identity with SEQ ID NO: 139.
  • the phaBCA operon has at least about 90% identity with SEQ ID NO: 139.
  • the phaBCA operon has at least about 95% identity with SEQ ID NO: 139.
  • the phaBCA operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 139.
  • the phaBCA operon comprises the sequence of SEQ ID NO: 139. In another embodiment, the phaBCA operon consists of the sequence of SEQ ID NO: 139. In one embodiment, the propionate catabolism gene cassette comprises prpE and phaBCA.
  • the propionate catabolism gene cassette comprises phaBCA.
  • the phaBCA operon has at least about 80% identity with SEQ ID NO: 102.
  • the phaBCA operon has at least about 90% identity with SEQ ID NO: 102.
  • the phaBCA operon has at least about 95% identity with SEQ ID NO: 102.
  • the phaBCA operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 102.
  • the phaBCA operon comprises the sequence of SEQ ID NO: 102. In another embodiment, the phaBCA operon consists of the sequence of SEQ ID NO: 102. In one embodiment, the propionate catabolism gene cassette comprises prpE and phaBCA.
  • the propionate catabolism gene cassette comprises prpE- phaBCA.
  • the prpE-phaBCA operon has at least about 80% identity with SEQ ID NO: 24.
  • the prpE-phaBCA operon has at least about 90% identity with SEQ ID NO: 24.
  • the prpE-phaBCA operon has at least about 95% identity with SEQ ID NO: 24.
  • the prpE-phaBCA operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 24.
  • the prpE- phaBCA operon comprises the sequence of SEQ ID NO: 24.
  • the prpE-phaBCA operon consists of the sequence of SEQ ID NO: 24.
  • the propionate catabolism gene cassette comprises prpE, pccB, accA1, mmcE, mutA, and mutB. Accordingly, in one embodiment, the prpE-pccB- accA1-mmcE-mutA-mutB operon has at least about 80% identity with a combination of SEQ ID NO: 37 and 31. In one embodiment, the prpE-pccB-accA1-mmcE-mutA-mutB operon has at least about 90% identity with a combination of SEQ ID NO: 37 and 31.
  • the prpE-pccB-accA1-mmcE-mutA-mutB operon has at least about 95% identity with a combination of SEQ ID NO: 37 and 31. In one embodiment, the prpE-pccB-accA1- mmcE-mutA-mutB operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a combination of SEQ ID NO: 37 and 31. In another embodiment, the prpE-pccB-accA1-mmcE-mutA-mutB operon comprises the sequence of a combination of SEQ ID NO: 37 and 31. In another embodiment, the prpE- pccB-accA1-mmcE-mutA-mutB operon consists of the sequence of a combination of SEQ ID NO: 37 and 31.
  • the propionate catabolism gene cassette comprises prpE, pccB, and accA1. Accordingly, in one embodiment, the prpE-pccB-accA1 operon has at least about 80% identity with SEQ ID NO: 37. In one embodiment, the prpE-pccB-accA1 operon has at least about 90% identity with SEQ ID NO: 37. In one embodiment, the prpE-pccB- accA1 operon has at least about 95% identity with SEQ ID NO: 37.
  • the prpE-pccB-accA1 operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37.
  • the prpE-pccB-accA1 operon comprises the sequence of SEQ ID NO: 37.
  • the prpE-pccB-accA1 operon consists of the sequence of SEQ ID NO: 37.
  • the propionate catabolism gene cassette comprises mmcE, mutA, and mutB. Accordingly, in one embodiment, the mmcE-mutA-mutB operon has at least about 80% identity with a combination of SEQ ID NO:31. In one embodiment, the mmcE- mutA-mutB operon has at least about 90% identity with a combination of SEQ ID NO: 31. In one embodiment, the -mmcE-mutA-mutB operon has at least about 95% identity with a combination of SEQ ID NO: 31.
  • the mmcE-mutA-mutB operon has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a combination of SEQ ID NO: 31.
  • the mmcE- mutA-mutB operon comprises the sequence of a combination of SEQ ID NO: 31.
  • the mmcE-mutA-mutB operon consists of the sequence of a combination of SEQ ID NO: 31.
  • the at least one gene encoding the at least one propionate catabolism enzyme is directly operably linked to a first promoter.
  • the at least one gene encoding the at least one propionate catabolism enzyme is indirectly operably linked to a first promoter.
  • the promoter is not operably linked with the at least one gene encoding the propionate catabolism enzyme in nature.
  • the at least one gene encoding the at least one propionate catabolism enzyme is expressed under the control of a constitutive promoter. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is expressed under the control of an inducible promoter. In some embodiments, the at least one gene encoding the at least one propionate catabolism enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions.
  • the at least one gene encoding the at least one propionate 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 propionate catabolism enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut.
  • a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions
  • expression of the at least one gene encoding the at least one propionate 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.
  • the at least one gene encoding the at least one propionate 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 propionate catabolism enzyme is located on a plasmid in the bacterial cell. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is located in the chromosome of the bacterial cell.
  • a native copy of the at least one gene encoding the at least one propionate catabolism enzyme is located in the chromosome of the bacterial cell, and at least one gene encoding at least one propionate catabolism enzyme from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the at least one gene encoding the at least one propionate catabolism enzyme is located on a plasmid in the bacterial cell, and at least one gene encoding the at least one propionate catabolism enzyme from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the at least one gene encoding the at least one propionate catabolism enzyme is located in the chromosome of the bacterial cell, and at least one gene encoding the at least one propionate catabolism enzyme from a different species of bacteria is located in the chromosome of the bacterial cell.
  • the at least one gene encoding the at least one propionate catabolism enzyme is expressed on a low-copy plasmid. In some embodiments, the at least one gene encoding the at least one propionate 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 propionate catabolism enzyme, thereby increasing the catabolism of propionate, propionic acid, propionyl CoA, methylmalonic acid, and/or methylmalonyl CoA.
  • a engineered bacterial cell comprising at least one gene encoding at least one propionate catabolism enzyme expressed on a high-copy plasmid does not increase propionate catabolism or decrease propionate, propionyl CoA, and/or methylmalonyl CoA levels as compared to a engineered bacterial cell comprising the same gene expressed on a low-copy plasmid in the absence of a heterologous importer of propionate and additional copies of a native importer of propionate. It has been surprisingly discovered that in some embodiments, the rate-limiting step of propionate catabolism is not expression of a propionate catabolism enzyme, but rather availability of propionate or propionyl CoA.
  • a transporter of propionate into the engineered bacterial cell there may be additional advantages to using a low-copy plasmid comprising the at least one gene encoding the at least one propionate catabolism enzyme in conjunction in order to enhance the stability of expression of the propionate catabolism enzyme, while maintaining high propionate catabolism and to reduce negative selection pressure on the transformed bacterium.
  • the importer of propionate is used in conjunction with a high-copy plasmid.
  • the engineered bacterial cell when the engineered bacterial cell expresses a heterologous PrpE enzyme, the engineered bacterial cell may further comprise a heterologous cobB gene (SEQ ID NO:114).
  • the cobB gene has at least about 80% identity with SEQ ID NO: 114. Accordingly, in one embodiment, the cobB gene has at least about 90% identity with SEQ ID NO: 114.
  • the cobB gene has at least about 95% identity with SEQ ID NO: 114. Accordingly, in one embodiment, the cobB gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 114. In another embodiment, the cobB gene comprises the sequence of SEQ ID NO: 114. In yet another embodiment the cobB gene consists of the sequence of SEQ ID NO: 114.
  • the at least one propionate catabolism enzyme is CobB. Accordingly, in one embodiment, CobB has at least about 113% identity with SEQ ID NO: 113. In one embodiment, CobB has at least about 90% identity with SEQ ID NO: 113. In another embodiment, CobB has at least about 95% identity with SEQ ID NO: 113.
  • CobB has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 113.
  • CobB comprises a sequence which encodes SEQ ID NO: 113.
  • CobB consists of a sequence which encodes SEQ ID NO: 113.
  • the engineered bacterial cell comprising a
  • heterologous cobB gene further comprises a genetic modification in the pka gene.
  • Pka a protein lysine acetyltransferase
  • PrpE Pr propionylated form
  • SEQ ID NO: 116 genetic modification of the pka gene which renders it functionally inactive enhances the ability of the bacterial cells to catabolize propionate.
  • Propionate transporters e.g., propionate importers
  • Propionate transporters may be expressed or modified in the bacteria in order to enhance propionate transport into the cell.
  • the transporter (importer) of propionate is expressed in the engineered bacterial cells
  • the bacterial cells import more propionate into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria comprising a heterologous gene encoding a transporter of propionate may be used to import propionate into the bacteria so that any gene encoding a propionate catabolism enzyme expressed in the organism can be used to treat diseases associated with the catabolism of propionate, such as organic acidurias (including PA and MMA) and vitamin B 12 deficiencies.
  • the bacterial cell comprises a heterologous gene encoding transporter of propionate.
  • the bacterial cell comprises a heterologous gene encoding a transporter of propionate and at least one heterologous gene encoding at least one propionate catabolism enzyme.
  • the disclosure provides a bacterial cell that comprises at least one heterologous gene encoding a propionate catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding a propionate transporter. In some embodiments, the disclosure provides a bacterial cell that comprises at least one heterologous gene encoding a transporter of propionate operably linked to the first promoter. In another embodiment, the disclosure provides a bacterial cell that comprises at least one heterologous gene encoding at least one propionate catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding of propionate operably linked to a second promoter.
  • 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. [0302] In one embodiment, the bacterial cell comprises at least one gene encoding a transporter of propionate from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one native gene encoding a transporter of propionate. In some embodiments, the at least one native gene encoding a transporter of propionate is not modified. In another embodiment, the bacterial cell comprises more than one copy of at least one native gene encoding a transporter of propionate.
  • the bacterial cell comprises a copy of at least one gene encoding a native importer of propionate, as well as at least one copy of at least one heterologous gene encoding a transporter of propionate from a different bacterial species.
  • the bacterial cell comprises at least one, two, three, four, five, or six copies of the at least one heterologous gene encoding a transporter of propionate.
  • the bacterial cell comprises multiple copies of the at least one heterologous gene encoding a transporter of propionate.
  • the importer of propionate is encoded by a transporter of propionate gene derived from a bacterial genus or species, including but not limited to, Bacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Lactobacillus, Pseudomonas, Salmonella, Staphylococcus, Bacillus subtilis, Campylobacter jejuni,
  • a transporter of propionate gene derived from a bacterial genus or species, including but not limited to, Bacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Lactobacillus, Pseudomonas, Salmonella, Staphylococcus, Bacillus subtilis, Campylobacter jejuni,
  • the bacteria is a Virgibacillus. In some embodiments, the bacterial is a Corynebacterium. In one embodiment, the bacteria is C. glutamicum. In another
  • the bacteria is C. diphtheria. In another embodiment, the bacteria is C.
  • the bacteria is S. coelicolor. In another embodiment, the bacteria is M. smegmatis. In another embodiment, the bacteria is N. farcinica. In another embodiment, the bacteria is E. coli. In another embodiment, the bacteria is B. subtilis.
  • the present disclosure further comprises genes encoding functional fragments of a transporter of propionate or functional variants of a transporter of propionate.
  • the term“functional fragment thereof” or“functional variant thereof” of a transporter of propionate relates to an element having qualitative biological activity in common with the wild-type importer of propionate from which the fragment or variant was derived.
  • a functional fragment or a functional variant of a mutated importer of propionate protein is one which retains essentially the same ability to import propionate into the bacterial cell as does the importer protein from which the functional fragment or functional variant was derived.
  • the engineered bacterial cell comprises at least one heterologous gene encoding a functional fragment of a transporter of propionate. In another embodiment, the engineered bacterial cell comprises at least one heterologous gene encoding a functional variant of a transporter of propionate.
  • Assays for testing the activity of a transporter of propionate, a transporter of propionate functional variant, or a transporter of propionate functional fragment are well known to one of ordinary skill in the art.
  • propionate import can be assessed by expressing the protein, functional variant, or fragment thereof, in a engineered bacterial cell that lacks an endogenous propionate importer.
  • Propionate import can also be assessed using mass spectrometry.
  • Propionate import can also be expressed using gas chromatography.
  • samples can be injected into a Perkin Elmer Autosystem XL Gas Chromatograph containing a Supelco packed column, and the analysis can be performed according to manufacturing instructions (see, for example, Supelco I (1998) Analyzing fatty acids by packed column gas chromatography, Bulletin 856B:2014).
  • samples can be analyzed for propionate import using high-pressure liquid chromatography (HPLC).
  • a computer-controlled Waters HPLC system equipped with a model 600 quaternary solvent delivery system, and a model 996 photodiode array detector, and components of the sample can be resolved with an Aminex HPX-87H (300 by 7.8 mm) organic acid analysis column (Bio-Rad Laboratories) (see, for example, Palacios et al., 2003, J. Bacteriol., 185(9):2802-2810).
  • genes encoding the importer of propionate have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the importer of propionate have been codon-optimized for use in Escherichia coli.
  • the present disclosure also encompasses genes encoding a transporter of propionate 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.
  • the at least one gene encoding a transporter of propionate is mutagenized; mutants exhibiting increased propionate transport are selected; and the mutagenized at least one gene encoding a transporter of propionate is isolated and inserted into the bacterial cell.
  • the at least one gene encoding a transporter of propionate is mutagenized; mutants exhibiting decreased propionate transport are selected; and the mutagenized at least one gene encoding a transporter of propionate is isolated and inserted into the bacterial cell.
  • the importer modifications described herein may be present on a plasmid or chromosome.
  • the propionate importer is MctC.
  • the mctC gene has at least about 80% identity to SEQ ID NO: 88. Accordingly, in one embodiment, the mctC gene has at least about 90% identity to SEQ ID NO: 88. Accordingly, in one embodiment, the mctC gene has at least about 95% identity to SEQ ID NO: 88.
  • the mctC gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 88.
  • the mctC gene comprises the sequence of SEQ ID NO: 88.
  • the mctC gene consists of the sequence of SEQ ID NO: 88.
  • the at least one propionate catabolism enzyme is MctC. Accordingly, in one embodiment, MctC has at least about 87% identity with SEQ ID NO: 87. In one embodiment, MctC has at least about 90% identity with SEQ ID NO: 87. In another embodiment, MctC has at least about 95% identity with SEQ ID NO: 87. Accordingly, in one embodiment, MctC has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 87. In another embodiment, MctC comprises a sequence which encodes SEQ ID NO: 87. In yet another embodiment, MctC consists of a sequence which encodes SEQ ID NO: 87.
  • the propionate importer is PutP_6.
  • the putP_6 gene has at least about 80% identity to SEQ ID NO: 90.
  • the putP_6 gene has at least about 90% identity to SEQ ID NO: 90. Accordingly, in one embodiment, the putP_6 gene has at least about 95% identity to SEQ ID NO: 90. Accordingly, in one embodiment, the putP_6 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 90. In another embodiment, the putP_6 gene comprises the sequence of SEQ ID NO: 90. In yet another embodiment the putP_6 gene consists of the sequence of SEQ ID NO: 90.
  • the at least one propionate catabolism enzyme is PutP_6. Accordingly, in one embodiment, PutP_6 has at least about 89% identity with SEQ ID NO: 89. In one embodiment, PutP_6 has at least about 90% identity with SEQ ID NO: 89. In another embodiment, PutP_6 has at least about 95% identity with SEQ ID NO: 89.
  • PutP 6 has at least about 85%, 86%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 89.
  • PutP_6 comprises a sequence which encodes SEQ ID NO: 89.
  • PutP_6 consists of a sequence which encodes SEQ ID NO: 89.
  • propionate importer genes are known to those of ordinary skill in the art. See, for example, Jolker et al., J. Bacteriol., 2009, 191(3):940-948.
  • the propionate importer comprises the mctBC genes from C. glutamicum.
  • the propionate importer comprises the dip0780 and dip0791 genes from C. diphtheria. In another embodiment, the propionate importer comprises the ce0909 and ce0910 genes from C. efficiens. In another embodiment, the propionate importer comprises the ce1091 and ce1092 genes from C. efficiens. In another embodiment, the propionate importer comprises the sco1822 and sco1823 genes from S. coelicolor. In another embodiment, the propionate importer comprises the sco1218 and sco1219 genes from S. coelicolor. In another embodiment, the propionate importer comprises the ce1091 and sco5827 genes from S. coelicolor.
  • the propionate importer comprises the m_5160, m_5161, m_5165, and m_5166 genes from M. smegmatis.
  • the propionate importer comprises the nfa 17930, nfa 17940, nfa 17950, and nfa 17960 genes from N. farcinica.
  • the propionate importer comprises the actP and yjcH genes from E. coli.
  • the propionate importer comprises the ywcB and ywcA genes from B. subtilis.
  • the bacterial cell comprises at least one heterologous gene encoding at least one propionate catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding a transporter of propionate.
  • the at least one heterologous gene encoding a transporter of propionate is operably linked to the first promoter. In other embodiments, the at least one heterologous gene encoding a transporter of propionate is operably linked to a second promoter. In one embodiment, the at least one gene encoding a transporter of propionate is directly operably linked to the second promoter. In another embodiment, the at least one gene encoding a transporter of propionate is indirectly operably linked to the second promoter.
  • expression of at least one gene encoding a transporter of propionate is controlled by a different promoter than the promoter that controls expression of the at least one gene encoding the at least one propionate catabolism enzyme. In some embodiments, expression of the at least one gene encoding a transporter of propionate is controlled by the same promoter that controls expression of the at least one propionate catabolism enzyme. In some embodiments, at least one gene encoding a transporter of propionate and the propionate catabolism enzyme are divergently transcribed from a promoter region. In some embodiments, expression of each of genes encoding the at least one gene encoding a transporter of propionate and the at least one gene encoding the at least one propionate catabolism enzyme is controlled by different promoters.
  • the promoter is not operably linked with the at least one gene encoding a transporter of propionate in nature.
  • the at least one gene encoding the importer of propionate is controlled by its native promoter.
  • the at least one gene encoding the importer of propionate is controlled by an inducible promoter.
  • the at least one gene encoding the importer of propionate is controlled by a promoter that is stronger than its native promoter.
  • the at least one gene encoding the importer of propionate is controlled by a constitutive promoter.
  • the promoter is an inducible promoter. Inducible promoters are described in more detail infra.
  • the at least one gene encoding a transporter of propionate is located on a plasmid in the bacterial cell. In another embodiment, the at least one gene encoding a transporter of propionate is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the at least one gene encoding a transporter of propionate is located in the chromosome of the bacterial cell, and a copy of at least one gene encoding a transporter of propionate from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the at least one gene encoding a transporter of a propionate is located on a plasmid in the bacterial cell, and a copy of at least one gene encoding a transporter of propionate 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 a transporter of propionate is located in the
  • chromosome of the bacterial cell and a copy of the at least one gene encoding a transporter of propionate from a different species of bacteria is located in the chromosome of the bacterial cell.
  • the at least one native gene encoding the importer in the bacterial cell is not modified, and one or more additional copies of the native importer are inserted into the genome.
  • the one or more additional copies of the native importer that is inserted into the genome are under the control of the same inducible promoter that controls expression of the at least one gene encoding the propionate catabolism enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the at least one propionate catabolism enzyme, or a constitutive promoter.
  • the at least one native gene encoding the importer is not modified, and one or more additional copies of the importer from a different bacterial species is inserted into the genome of the bacterial cell.
  • the one or more additional copies of the importer inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the at least one gene encoding the propionate catabolism enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the at least one gene encoding the at least one propionate catabolism enzyme, or a constitutive promoter.
  • the bacterial cells import 10% more propionate into the bacterial cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the importer of propionate is expressed in the engineered bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more propionate into the bacterial cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the bacterial cells import two-fold more propionate into the cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the importer of propionate is expressed in the engineered bacterial cells, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more propionate into the cell when the importer is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • Succinate export in bacteria is normally active under anaerobic conditions.
  • the export of succinate is mediated by proteins well known to those of skill in the art.
  • a succinate exporter in Corynebacterium glutamicum is known as SucE1.
  • SucE1 is a membrane protein belonging to the aspartate:alanine exchanger (AAE) family (see, for example, Fukui et al., 2011, J. Bacteriol., 154(1):25-34).
  • AAE aspartate:alanine exchanger
  • the DcuC succinate exporter from E. coli has also been identified (see, for example, Cheng et al., 2013, J. Biomed. Res. Int., 2013:ID 538790).
  • Succinate transporters e.g., succinate exporters
  • succinate exporters may be expressed or modified in the bacteria in order to enhance succinate export out of the cell.
  • the bacterial cells export more succinate outside of the cell when the exporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the bacterial cell comprises a heterologous gene encoding an exporter of succinate.
  • the bacterial cell comprises a heterologous gene encoding an exporter of succinate and at least one heterologous gene or gene cassette encoding at least one propionate catabolism enzyme.
  • the disclosure provides a bacterial cell that comprises at least one heterologous gene or gene cassette encoding a propionate catabolism enzyme or enzymes operably linked to a first promoter and at least one heterologous gene encoding an exporter of succinate.
  • the at least one heterologous gene encoding an exporter of succinate is operably linked to the first promoter.
  • the at least one heterologous gene encoding the at least one propionate catabolism enzyme operably is linked to a first promoter
  • the heterologous gene encoding an exporter of succinate is operably linked to a second promoter.
  • the first promoter and the second promoter are separate copies of the same promoter.
  • the first promoter and the second promoter are different promoters.
  • the bacterial cell comprises at least one gene encoding an exporter of succinate from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises at least one native gene encoding an exporter of succinate.
  • the at least one native gene encoding an exporter of succinate is not modified.
  • the bacterial cell comprises more than one copy of at least one native gene encoding an exporter of succinate.
  • the bacterial cell comprises a copy of at least one gene encoding a native exporter of succinate, as well as at least one copy of at least one heterologous gene encoding an exporter of succinate from a different bacterial species.
  • the bacterial cell comprises at least one, two, three, four, five, or six copies of the at least one heterologous genes encoding an exporter of succinate. In one embodiment, the bacterial cell comprises multiple copies of the at least one heterologous gene encoding an exporter of succinate.
  • the exporter of succinate is encoded by an exporter of succinate gene derived from a bacterial genus or species, including but not limited to, Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, and Mannheimia succiniciproducens, Escherichia coli, Corynebacterium glutamicum, Salmonella
  • the exporter of succinate is derived from Corynebacterium. In one
  • the exporter of succinate is derived from C. glutamicum. In another embodiment, the exporter of succinate is from Vibrio cholerae. In another embodiment, the exporter of succinate is from E. coli. In another embodiment, the exporter of succinate is from Bacillus subtilis.
  • the present disclosure further comprises genes encoding functional fragments of an exporter of succinate or functional variants of an exporter of succinate.
  • the term“functional fragment thereof” or“functional variant thereof” of an exporter of succinate relates to an element having qualitative biological activity in common with the wild-type exporter of succinate from which the fragment or variant was derived.
  • a functional fragment or a functional variant of a mutated exporter of succinate protein is one which retains essentially the same ability to import succinate into the bacterial cell as does the exporter protein from which the functional fragment or functional variant was derived.
  • the engineered bacterial cell comprises at least one
  • the engineered bacterial cell comprises at least one heterologous gene encoding a functional variant of an exporter of succinate.
  • the genetically engineered bacteria further comprise a mutation or deletion in one or more succinate importers, e.g., Dct, DctC, ybhI or ydjN.
  • succinate dehydrogenase SUCDH
  • such mutations may decrease intracellular succinate concentrations and increase the flux through propionate catabolism pathways.
  • succinate export can be assessed by expressing the protein, functional variant, or fragment thereof, in a engineered bacterial cell that lacks an endogenous succinate exporter and assessing succinate levels in the media after expression of the protein.
  • Methods for measuring succinate export are well known to one of ordinary skill in the art. For example, see Fukui et al., J. Biotechnol., 154(1):25-34, 2011.
  • the genes encoding the exporter of succinate have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the exporter of succinate have been codon-optimized for use in Escherichia coli.
  • the present disclosure also encompasses genes encoding an exporter of succinate 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.
  • the at least one gene encoding an exporter of succinate is mutagenized; mutants exhibiting increased succinate transport are selected; and the mutagenized at least one gene encoding an exporter of succinate is isolated and inserted into the bacterial cell.
  • the at least one gene encoding an exporter of succinate is mutagenized; mutants exhibiting decreased succinate transport are selected; and the mutagenized at least one gene encoding an exporter of succinate is isolated and inserted into the bacterial cell.
  • the exporter modifications described herein may be present on a plasmid or chromosome.
  • the succinate exporter is DcuC.
  • the dcuC gene has at least about 80% identity to SEQ ID NO: 49. Accordingly, in one embodiment, the dcuC gene has at least about 90% identity to SEQ ID NO: 49. Accordingly, in one embodiment, the dcuC gene has at least about 95% identity to SEQ ID NO: 49.
  • the dcuC gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49.
  • the dcuC gene comprises the sequence of SEQ ID NO: 49.
  • the dcuC gene consists of the sequence of SEQ ID NO:70.
  • the at least one propionate catabolism enzyme is DcuC. Accordingly, in one embodiment, DcuC has at least about 89% identity with SEQ ID NO: 129. In one embodiment, DcuC has at least about 90% identity with SEQ ID NO: 129. In another embodiment, DcuC has at least about 95% identity with SEQ ID NO: 129.
  • DcuC has at least about 85%, 86%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 129.
  • DcuC comprises a sequence which encodes SEQ ID NO: 129.
  • DcuC consists of a sequence which encodes SEQ ID NO: 129.
  • the succinate exporter is DcuC.
  • the dcuC gene has at least about 80% identity to SEQ ID NO: 118. Accordingly, in one embodiment, the dcuC gene has at least about 90% identity to SEQ ID NO: 118.
  • the dcuC gene has at least about 95% identity to SEQ ID NO: 118. Accordingly, in one embodiment, the dcuC gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 118. In another embodiment, the dcuC gene comprises the sequence of SEQ ID NO: 118. In yet another embodiment the dcuC gene consists of the sequence of SEQ ID NO: 118.
  • the at least one propionate catabolism enzyme is DcuC. Accordingly, in one embodiment, DcuC has at least about 89% identity with SEQ ID NO: 117. In one embodiment, DcuC has at least about 90% identity with SEQ ID NO: 117. In another embodiment, DcuC has at least about 95% identity with SEQ ID NO: 117.
  • DcuC has at least about 85%, 86%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 117.
  • DcuC comprises a sequence which encodes SEQ ID NO: 117.
  • DcuC consists of a sequence which encodes SEQ ID NO: 117.
  • the succinate exporter is SucE1.
  • the sucE1 gene has at least about 80% identity to SEQ ID NO: 46. Accordingly, in one embodiment, the sucE1 gene has at least about 90% identity to SEQ ID NO: 46.
  • the sucE1 gene has at least about 95% identity to SEQ ID NO: 46. Accordingly, in one embodiment, the sucE1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46. In another embodiment, the sucE1 gene comprises the sequence of SEQ ID NO: 46. In yet another embodiment the sucE1 gene consists of the sequence of SEQ ID NO: 46.
  • the succinate exporter is SucE1.
  • the sucE1 gene has at least about 80% identity to SEQ ID NO: 120. Accordingly, in one embodiment, the sucE1 gene has at least about 90% identity to SEQ ID NO: 120.
  • the sucE1 gene has at least about 95% identity to SEQ ID NO: 120. Accordingly, in one embodiment, the sucE1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 120. In another embodiment, the sucE1 gene comprises the sequence of SEQ ID NO: 120. In yet another embodiment the sucE1 gene consists of the sequence of SEQ ID NO: 120.
  • the at least one succinate exporter is sucE1. Accordingly, in one embodiment, sucE1 has at least about 89% identity with SEQ ID NO: 128. In one embodiment, sucE1 has at least about 90% identity with SEQ ID NO: 128. In another embodiment, sucE1 has at least about 95% identity with SEQ ID NO: 128. Accordingly, in one embodiment, sucE1 has at least about 85%, 86%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 128. In another embodiment, sucE1 comprises a sequence which encodes SEQ ID NO: 128.
  • sucE1 consists of a sequence which encodes SEQ ID NO: 128. In another embodiment, the sucE1 has at least about 89% identity with SEQ ID NO: 119. In one embodiment, sucE1 has at least about 90% identity with SEQ ID NO: 119. In another embodiment, sucE1 has at least about 95% identity with SEQ ID NO: 119. Accordingly, in one embodiment, sucE1 has at least about 85%, 86%, 89%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 119. In another embodiment, sucE1 comprises a sequence which encodes SEQ ID NO: 119. In yet another embodiment, sucE1 consists of a sequence which encodes SEQ ID NO: 119.
  • the bacterial cell comprises at least one heterologous gene encoding at least one propionate catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding an exporter of succinate.
  • the at least one heterologous gene encoding an exporter of succinate is operably linked to the first promoter.
  • the at least one heterologous gene encoding an exporter of succinate is operably linked to a second promoter.
  • the at least one gene encoding an exporter of succinate is directly operably linked to the second promoter.
  • the at least one gene encoding an exporter of succinate is indirectly operably linked to the second promoter.
  • expression of at least one gene encoding an exporter of succinate is controlled by a different promoter than the promoter that controls expression of the at least one gene encoding the at least one propionate catabolism enzyme.
  • expression of the at least one gene encoding an exporter of succinate is controlled by the same promoter that controls expression of the at least one propionate catabolism enzyme.
  • at least one gene encoding an exporter of succinate and the propionate catabolism enzyme are divergently transcribed from a promoter region.
  • expression of each of genes encoding the at least one gene encoding an exporter of succinate and the at least one gene encoding the at least one propionate catabolism enzyme is controlled by different promoters.
  • the promoter is not operably linked with the at least one gene encoding an exporter of succinate in nature.
  • the at least one gene encoding the exporter of succinate is controlled by its native promoter.
  • the at least one gene encoding the exporter of succinate is controlled by an inducible promoter. In some embodiments, the at least one gene encoding the exporter of succinate is controlled by a promoter that is stronger than its native promoter. In some embodiments, the at least one gene encoding the exporter of succinate is controlled by a constitutive promoter.
  • the promoter is an inducible promoter. Inducible promoters are described in more detail infra.
  • the at least one gene encoding an exporter of succinate is located on a plasmid in the bacterial cell.
  • the at least one gene encoding an exporter of succinate is located in the chromosome of the bacterial cell.
  • a native copy of the at least one gene encoding an exporter of succinate is located in the chromosome of the bacterial cell, and a copy of at least one gene encoding an exporter of succinate from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the at least one gene encoding an exporter of a succinate is located on a plasmid in the bacterial cell, and a copy of at least one gene encoding an exporter of succinate from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the at least one gene encoding an exporter of succinate is located in the chromosome of the bacterial cell, and a copy of the at least one gene encoding an exporter of succinate from a different species of bacteria is located in the chromosome of the bacterial cell.
  • the at least one native gene encoding the exporter in the bacterial cell is not modified, and one or more additional copies of the native exporter are inserted into the genome.
  • the one or more additional copies of the native exporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the at least one gene encoding the propionate catabolism enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the at least one propionate catabolism enzyme, or a constitutive promoter.
  • the at least one native gene encoding the exporter is not modified, and one or more additional copies of the exporter from a different bacterial species is inserted into the genome of the bacterial cell.
  • the one or more additional copies of the exporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the at least one gene encoding the propionate catabolism enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the at least one gene encoding the at least one propionate catabolism enzyme, or a constitutive promoter.
  • the bacterial cells when the exporter of succinate is expressed in the engineered bacterial cells, the bacterial cells export 10% more succinate out of the bacterial cell when the exporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the exporter of succinate is expressed in the engineered bacterial cells, the bacterial cells export 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more succinate out of the bacterial cell when the exporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the bacterial cells when the exporter of succinate is expressed in the engineered bacterial cells, the bacterial cells export two-fold more succinate out of the cell when the exporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the exporter of succinate is expressed in the engineered bacterial cells, the bacterial cells export three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more succinate out of the cell when the exporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • Essential Genes and Auxotrophs are essential Genes and Auxotrophs
  • essential gene refers to a gene which is necessary to for cell growth and/or survival.
  • Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).
  • 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.
  • 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.
  • any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth.
  • the essential gene is an oligonucleotide synthesis gene, for example, thyA.
  • the essential gene is a cell wall synthesis gene, for example, dapA.
  • the essential gene is an amino acid gene, for example, serA or MetA.
  • Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria.
  • 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 oli onucleotide s nthesis, amino acid s nthesis, and cell wall s nthesis.
  • 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.
  • thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death.
  • the thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003).
  • 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.
  • 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.
  • 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).
  • Diaminopimelic acid is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971).
  • 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.
  • 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).
  • the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene.
  • the uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995).
  • a uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies.
  • 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).
  • an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph.
  • the genetically engineered bacteria comprise a deletion or mutation in two or more genes required for cell survival and/or growth.
  • essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR,
  • 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
  • the SLiDE bacterial cell comprises a mutation in an essential gene.
  • the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk.
  • 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
  • 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.
  • 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.
  • the genetically engineered bacterium is complemented by a ligand.
  • 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.
  • bacterial cells comprising mutations in metG 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 are complemented by benzothiazole, indole or 2-aminobenzothiazole.
  • Bacterial cells comprising mutations in pheS are
  • Bacterial cells comprising mutations in tyrS are complemented by benzothiazole or 2- aminobenzothiazole.
  • Bacterial cells comprising mutations in adk are complemented by benzothiazole or indole.
  • the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand.
  • the bacterial cell comprises mutations in two essential genes.
  • the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C).
  • the bacterial cell comprises mutations in three essential genes.
  • 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).
  • the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.
  • 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.
  • 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).
  • 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).
  • 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 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein or rescue an auxotroph.
  • the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.
  • the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a T7 polymerase-regulated genetic regulatory circuit.
  • 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.
  • FNR fumarate and nitrate reductase regulator
  • lysY an inhibitory factor
  • 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.
  • 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.
  • the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a protease-regulated genetic regulatory circuit.
  • 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.
  • FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the payload is not expressed.
  • 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.
  • the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a repressor-regulated genetic regulatory circuit.
  • 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.
  • 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.
  • FNR dimerizes and binds the FNR- responsive promoter the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.
  • repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, LacI, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).
  • the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a regulatory RNA-regulated genetic regulatory circuit.
  • 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.
  • FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated.
  • FNR dimerizes and binds the FNR-responsive promoter the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.
  • the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a CRISPR-regulated genetic regulatory circuit.
  • 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.
  • FNR does not bind the FNR-responsive promoter
  • the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed.
  • FNR dimerizes and binds the FNR-responsive promoter the guide RNA is expressed, the repressor is not expressed, and the payload is expressed.
  • the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a recombinase-regulated genetic regulatory circuit.
  • 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’).
  • 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.
  • 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.
  • 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.
  • 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’).
  • FNR does not bind the FNR-responsive promoter
  • the recombinase is not expressed
  • the T7 polymerase gene remains in the 3’ to 5’ orientation
  • the payload is not expressed.
  • 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.
  • 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.
  • 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.
  • 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.
  • the kill switch is activated to kill the bacteria after a period of time following expression of the propionate catabolism enzyme cassette(s) and/or gene(s) present in the engineered bacteria.
  • 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).
  • the bacteria may be engineered to die after the bacteria has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject).
  • Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination.
  • the switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species.
  • 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.
  • an AND riboregulator switch is activated by tetracycline, isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010).
  • 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).
  • 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).
  • 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
  • a toxin is produced once an environmental condition no longer exists or an external signal is ceased (i.e., a repression-based kill switch).
  • 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.
  • 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.
  • the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then
  • constitutive expression of the bacterial toxin kills the genetically engineered bacterium.
  • the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the engineered bacterial cell is no longer viable.
  • 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.
  • the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase.
  • the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence.
  • the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase.
  • the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium.
  • 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.
  • 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.
  • the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence.
  • the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence.
  • the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase.
  • the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase.
  • the genetically engineered bacterium is killed by the bacterial toxin.
  • the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition.
  • the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium.
  • 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.
  • 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.
  • 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.
  • the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase.
  • 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.
  • the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase.
  • the first excision enzyme excises a first essential gene.
  • the programmed engineered bacterial cell is not viable after the first essential gene is excised.
  • 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.
  • the first excision enzyme is Xis1. In one embodiment, the first excision enzyme is Xis2. In one embodiment, the first excision enzyme is Xis1, and the second excision enzyme is Xis2.
  • 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.
  • the recombinase can be a recombinase selected from the group consisting of: BxbI, PhiC31, TP901, BxbI, PhiC31, TP901, HK022, HP1, R4, Int1, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, Int10, Int11, Int12, Int13, Int14, Int15, Int16, Int17, Int18, Int19, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.
  • a toxin is produced in the presence of an environmental factor or signal.
  • 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.
  • 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 araBAD promoter.
  • the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene.
  • the toxin gene is repressed in the presence of arabinose or other sugar.
  • 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.
  • heterologous genes are directly or indirectly under the control of the araBAD promoter.
  • 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.
  • Arabinose inducible promoters are known in the art, including P ara , P araB , P araC , and P araBAD .
  • the arabinose inducible promoter is from E. coli.
  • the P araC promoter and the P araBAD promoter operate as a bidirectional promoter, with the P araBAD promoter controlling expression of a heterologous gene(s) in one direction, and the P araC (in close proximity to, and on the opposite strand from the P araBAD promoter), controlling expression of a heterologous gene(s) in the other direction.
  • the P araC promoter and the P araBAD promoter operate as a bidirectional promoter, with the P araBAD promoter controlling expression of a heterologous gene(s) in one direction, and the P araC (in close proximity to, and on the opposite strand from the P araBAD promoter), controlling expression of a heterologous gene(s) in the other direction.
  • the P araC promoter controlling expression of a heterologous gene(s) in one direction
  • the P araC in close proximity to, and on the opposite strand from the
  • the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P araBAD promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a P araC promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (P TetR ).
  • TetR Tetracycline Repressor Protein
  • the AraC transcription factor activates the P araBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin.
  • AraC suppresses transcription from the P araBAD promoter and no TetR protein is expressed.
  • 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.
  • the araC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore
  • the engineered bacterial cell further comprises an antitoxin under the control of a constitutive promoter.
  • the toxin 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.
  • TetR protein 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.
  • the engineered bacterial cell further comprises an antitoxin under the control of the P araBAD promoter.
  • an antitoxin under the control of the P araBAD promoter.
  • 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.
  • 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.
  • the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell (and required for survival), and a P araC promoter operably linked to a heterologous gene encoding AraC transcription factor.
  • a P araBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell (and required for survival)
  • a P araC promoter operably linked to a heterologous gene encoding AraC transcription factor.
  • the AraC transcription factor activates the P araBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the engineered bacterial cell to survive.
  • the engineered bacterial cell dies in the absence of arabinose.
  • the sequence of P araBAD 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.
  • sequence of P araBAD 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.
  • the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin.
  • the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin.
  • the short-lived anti-toxin begins to decay.
  • the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it.
  • the engineered bacteria of the present disclosure may further comprise the gene(s) encoding the
  • the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, Ibs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin I47, microcin M, colicin A, colicin E1, colicin K, colicin N, colicin U, colicin B, colicin I
  • the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccE CTD , MccF, Cai, ImmE1, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.
  • an anti-lysin Sok
  • RNAII IstR, RdlD, Kis, SymR, MazE,
  • the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.
  • 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.
  • the heterologous reporter gene is a fluorescence gene.
  • the fluorescence gene encodes a green fluorescence protein (GFP).
  • 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 al., Science Translational Medicine, 7(289):1-12, 2015, the entire contents of which are expressly incorporated herein by reference).
  • 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.
  • the disclosure provides an isolated plasmid further comprising a third nucleic acid encoding a third payload operably linked to a third inducible promoter.
  • 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.
  • the first, second, third, fourth, fifth, sixth, etc“payload(s)” can be a propionate catabolism enzyme, a propionate transporter, a propionate binding protein, or other sequence described herein.
  • the nucleic acid encoding the first payload and the nucleic acid encoding the second payload are operably linked to the first inducible promoter.
  • 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.
  • 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.
  • 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
  • the nucleic acid encoding te third payload is operably linked to a third inducible promoter.
  • the first, second, and third inducible promoters are separate copies of the same inducible promoter.
  • the first inducible promoter, the second inducible promoter, and the third inducible promoter are different inducible promoters.
  • 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.
  • 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.
  • FNR fumarate and nitrate reduction regulator
  • 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.
  • 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.
  • the heterologous gene encoding a propionate catabolism enzyme is operably linked to a constitutive promoter.
  • the constitutive promoter is a lac promoter.
  • the constitutive promoter is a tet promoter.
  • the constitutive promoter is a constitutive
  • the constitutive promoter is a constitutive Escherichia coli ⁇ 32 promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli ⁇ 70 promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis ⁇ A promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis ⁇ B 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.
  • the plasmid further comprises a heterologous gene encoding a propionate transporter, a propionate binding protein, and/or a kill switch construct, which may be operably linked to a constitutive promoter or an inducible promoter.
  • the isolated plasmid comprises at least one
  • heterologous propionate 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.
  • the isolated plasmid comprises at least one heterologous gene encoding a propionate catabolism enzyme operably linked to a first inducible promoter; a heterologous gene encoding a TetR protein and an anti-toxin 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.
  • a first nucleic acid encoding a propionate catabolism enzyme comprises a prpE and/or a Pha gene.
  • a first nucleic acid encoding a propionate catabolism enzyme is a Pha gene or a Pha operon, e.g. prpE-phaB- phaC-phaA.
  • the prpE gene or Pha gene or Pha operon is coexpressed with an additional propionate catabolism gene or gene cassette, e.g. a MMCA cassette and/or a 2MC cassette described herein.
  • a gene encoding a succinate exporter e.g., SucE1 and/or DcuC
  • a priopionate importer is further expressed.
  • a first nucleic acid encoding a propionate catabolism enzyme comprises a prpE and/or a MMCA pathway gene.
  • a first nucleic acid encoding a propionate catabolism enzyme is a prpE and/or a MMCA pathway gene or a MMCA pathway operon, e.g. prpE-accA1-pccB-mmcE-mutA-mutB or prpE- accA1-pccB or mmcE-mutA-mutB.
  • the prpE and/or a MMCA pathway gene or a MMCA pathway operon is coexpressed with an additional propionate catabolism gene or gene cassette, e.g. a Pha cassette and/or a 2MC cassette described herein.
  • an additional propionate catabolism gene or gene cassette e.g. a Pha cassette and/or a 2MC cassette described herein.
  • a gene encoding a succinate exporter e.g., SucE1 and/or DcuC
  • a priopionate importer is further expressed.
  • a first nucleic acid encoding a propionate catabolism enzyme comprises a prpE and/or a 2MC pathway gene.
  • a first nucleic acid encoding a propionate catabolism enzyme is a prpE and/or a 2MC pathway gene or a 2MC pathway operon, e.g. prpB-prpC-prpD-prpE or prpB-prpC-prpD.
  • the prpE and/or a 2MC pathway gene or a 2MC pathway operon is
  • a gene encoding a succinate exporter e.g., SucE1 and/or DcuC
  • a priopionate importer is further expressed.
  • the plasmid is a high-copy plasmid. In another embodiment, the plasmid is a low-copy plasmid.
  • 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.
  • the bacterial cell further comprises a genetic mutation in an endogenous gene encoding a lysine acetyltransferase, e.g. pka, which propionylates and inactivates prpE.
  • the bacterial cell further comprises a genetic mutation which reduces export of propionate and/or its metabolites from the bacterial cell.
  • the bacterial cell further comprises a genetic mutation in an endogenous gene encoding a propionate biosynthesis gene, wherein the genetic mutation reduces biosynthesis of propionate and one or more of its metabolites in the bacterial cell.
  • the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions.
  • MOAs mechanisms of action
  • insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in FIG.32.
  • the genetically engineered bacteria may include four copies of a propionate catabolism gene or propionate catabolism gene cassette, or four copies of a propionate catabolism gene inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ.
  • the genetically engineered bacteria may include one or more copies of a propionate 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 propionate 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 propionate catabolism gene or gene cassette inserted at one or more different insertion sites.
  • a propionate 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 propionate 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 propionate catabolism gene or gene cassette inserted at one or more
  • the genetically engineered bacteria comprise one or more of: (1) one or more gene(s) and/or gene cassettes encoding one or more propionate 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 propionate and/or one or more of its metabolites, including methylmalonic acid, 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 propionate catabolism enzyme(s) for secretion and extracellular degradation of propionate and/or one or more of its metabolites, (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),
  • the genetically engineered bacteria comprise two or more different pathway cassettes or operons comprising propionate catabolism enzymes. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more propionate catabolism enzymes selected from PrpE, AccA1, PccB, MmcE, MutA, and MutB, and combinations thereof.
  • the genetically engineered bacteria comprise gene sequence(s) comprising two or more copies of any genes selected from prpE, accA1, pccB, mmcE, mutA, and mutB. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more propionate catabolism enzymes selected from PrpE, PhaB, PhaC, and PhaA, and combinations thereof. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) comprising two or more copies of any genes selected from prpE, phaB, phaC, and phaA.
  • the genetically engineered bacteria comprise gene sequence encoding one or more propionate catabolism enzymes selected from PrpB, PrpC, PrpD, and PrpE, and combinations thereof. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) comprising two or more copies of any genes selected from prpB-prpC, prpD, and prpE.
  • Non limiting examples of combinations include genetically engineered bacteria comprising one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in combination with one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA).
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • PHA pathway operon(s) e.g., prpE-phaB-phaC-phaA
  • the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accA1- pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in combination with one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE).
  • MMCA pathway operon(s) e.g., prpE-accA1- pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB), one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA).
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • PHA pathway operon(s) e.g.,
  • the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA).
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • PHA pathway operon(s) e.g., prpE-phaB-phaC-phaA
  • the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB- prpC-prpD-prpE), and one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB- mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB).
  • 2MC pathway operon(s) e.g., prpB- prpC-prpD-prpE
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB- mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB.
  • Non limiting examples of combinations include genetically engineered bacteria comprising one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE- mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in combination with one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and in combination with one or more cassettes comprising matB.
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE- mutA-mutB
  • prpE-accA1-pccB and mmcE-mutA-mutB e.g., prpE-phaB-phaC-phaA
  • the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accA1- pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in combination with one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE) and in combination with one or more cassettes comprising matB.
  • MMCA pathway operon(s) e.g., prpE-accA1- pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE- accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB), one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and in combination with one or more cassettes comprising matB.
  • MMCA pathway operon(s) e.g., prpE- accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD- prpE), and one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and in combination with one or more cassettes comprising MatB.
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD- prpE
  • PHA pathway operon(s) e.g., prpE-phaB-phaC-phaA
  • the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) and in combination with one or more cassettes comprising matB.
  • Any of the combinations described above comprising matB may or may not comprise prpE, e.g., may comprise matB in lieu of prpE.
  • the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes and one or more gene(s) or gene cassette(s) encoding one or more propionate transporters (importers), such as any of the propionate transporters described herein and otherwise known in the art.
  • the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • Non limiting examples of combinations include genetically engineered bacteria comprising one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA- mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in combination with one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA- mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • PHA pathway operon(s) e.g., prpE-phaB-phaC-phaA
  • the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in combination with one or more 2MC pathway operon(s) (e.g., prpB- prpC-prpD-prpE) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • 2MC pathway operon(s) e.g., prpB- prpC-prpD-prpE
  • the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE- accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB), one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • MMCA pathway operon(s) e.g., prpE- accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more PHA pathway operon(s) (e.g., prpE- phaB-phaC-phaA) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • PHA pathway operon(s) e.g., prpE- phaB-phaC-phaA
  • gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), and one or more MMCA pathway operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC.
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucE1 and/or dcuC, e.g., as described supra, may comprise one or more gene(s) or gene cassette(s) comprising matB or matB may be substituted in lieu of prpE.
  • the engineered bacterium may also comprise gene sequence(s) encoding one or more propionate transporters.
  • the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes and one or more genetic modifications that reduce or decrease succinate import into the bacterial cell, such as any of the genetic modifications described herein and otherwise known in the art.
  • the engineered bacterium may further comprise gene sequence(s) encoding one or more propionate transporters.
  • the engineered bacterium may further comprise gene sequence encoding one or more succinate exporters.
  • the engineered bacterium comprises one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes, one or more genetic modifications that reduce or decrease succinate import into the bacterial cell, and gene sequence(s) encoding one or more propionate transporters.
  • the engineered bacterium comprises one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes, one or more genetic modifications that reduce or decrease succinate import into the bacterial cell, and gene sequence(s) encoding one or more succinate exporters.
  • the engineered bacterium comprises one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes, one or more genetic modifications that reduce or decrease succinate import into the bacterial cell, gene sequence(s) encoding one or more propionate transporters, and gene sequence(s) encoding one or more succinate exporters.
  • 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).
  • the genetically engineered bacteria may encode one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and one or more additional gene(s) or gene cassette(s) encoding one or more of phaA.
  • the genetically engineered bacteria may one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and one or more additional gene(s) or gene cassette(s) encoding one or more of prpE and/or phaB and/or phaC and/or phaA.
  • PHA pathway operon(s) e.g., prpE-phaB-phaC-phaA
  • additional gene(s) or gene cassette(s) encoding one or more of prpE and/or phaB and/or phaC and/or phaA.
  • the genetically engineered bacteria may encode one or more MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE- accA1-pccB and mmcE-mutA-mutB) and one or more additional gene(s) or gene cassette(s) encoding one or more of prpE and/or accA1 and/or opccB and/or mmcE and/or mutA and/or mutB.
  • MMCA pathway operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE- accA1-pccB and mmcE-mutA-mutB
  • additional gene(s) or gene cassette(s) encoding one or more of prpE and/or accA1 and/or opccB and/or mmcE and/or mutA and
  • the genetically engineered bacteria may one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE) and one or more additional gene(s) or gene cassette(s) encoding prpB and/or prpC and/or prpD and/or prpE).
  • 2MC pathway operon(s) e.g., prpB-prpC-prpD-prpE
  • additional gene(s) or gene cassette(s) encoding prpB and/or prpC and/or prpD and/or prpE.
  • each gene from a propionate catabolism pathway described herein can be expressed individually, each under control of a separate (same or different) promoter.
  • a separate (same or different) promoter e.g., PHA, MMCA, and/or 2MC
  • prpE and/or phaB and/or phaC and/or phaA can be expressed individually, each under control of a separate (same or different) promoter.
  • prpE and/or accA1 and/or opccB and/or mmcE and/or mutA and/or mutB can be expressed individually, each under control of a separate (same or different) promoter.
  • prpB and/or prpC and/or prpD and/or prpE can be expressed individually, each under control of a separate (same or different) promoter.
  • each gene from a propionate catabolism pathway described herein, e.g., a matB comprising pathway (e.g., matA, mmcE, mutA and mutB, and/or MatB, Acc1A, and PccB, (e.g., with PrpE)) can be expressed individually, each under control of a separate (same or different) promoter.
  • 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.
  • the genetically engineered bacteria may encode one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA), in phaC comes first or phaB comes first, or prpE comes first or phaA comes first.
  • the genetically engineered bacteria may encode one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA), in which that phaC comes second or phaB comes second, or prpE comes second or phaA comes second.
  • the genetically engineered bacteria may encode one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA), in which phaC comes third or phaB comes third, or prpE comes third or phaA comes third.
  • PHA pathway operon(s) e.g., prpE-phaB-phaC-phaA
  • the genetically engineered bacteria may encode one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), in which prpB comes first or prpC comes first or prpD comes first or prpE comes first.
  • the genetically engineered bacteria may encode one or more 2MC pathway operon(s) (e.g., prpB- prpC-prpD-prpE), in which prpB comes second or prpC comes second or prpD comes second or prpE comes second.
  • the genetically engineered bacteria may encode one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), in which prpB comes third or prpC comes third or prpD comes third or prpE comes third.
  • the genetically engineered bacteria may encode one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE), in which prpB comes fourth or prpC comes fourth or prpD comes fourth or prpE comes fourth.
  • the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in which prpE comes first or accA1 comes first or pccB comes first or mmcE comes first or mutA comes first or mutB comes first.
  • MMCA operon(s) e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE- accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in which prpE comes second or accA1 comes second or pccB comes second or mmcE comes second or mutA comes second or mutB comes second.
  • MMCA operon(s) e.g., prpE- accA1-pccB-mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-accA1-pccB- mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB) in which prpE comes third or accA1 comes third or pccB comes third or mmcE comes third or mutA comes third or mutB comes third.
  • MMCA operon(s) e.g., prpE-accA1-pccB- mmcE-mutA-mutB, or prpE-accA1-pccB and mmcE-mutA-mutB
  • the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-accA1-pccB-mmcE-mutA-mutB, or prpE- accA1-pccB and mmcE-mutA-mutB) in which prpE comes fourth, fifth or sixth or accA1 comes fourth, fifth or sixth or pccB comes fourth, fifth or sixth or mmcE comes fourth, fifth or sixth or mutA comes fourth, fifth or sixth or mutB comes fourth, fifth or sixth.
  • matB comes first, second, third, fourth, fifith, or sixth in a gene cassette comprising matB.
  • any one or more the genes can be operably linked to a diercetly 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.
  • 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 propionate catabolism enzyme within a cassette.
  • the genetically engineered bacteria further comprise mutations or deletions, e.g., in pka, succinate importers or propionate exporters, and an auxotrophy.
  • the genetically engineered bacteria also comprise a plasmid that has been modified to create a host-plasmid mutual dependency.
  • the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015).
  • 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).
  • the GeneGuard plasmid is stable for at least one- hundred generations without antibiotic selection.
  • 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.
  • the mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies).
  • the genetically engineered bacteria comprise a GeneGuard plasmid.
  • the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches.
  • the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies.
  • the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.
  • the vector comprises a conditional origin of replication.
  • the conditional origin of replication is a R6K or ColE2- P9.
  • the host cell expresses the replication initiator protein pi.
  • the host cell expresses the replication initiator protein RepA.
  • 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.
  • 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.
  • the vector comprises a gene encoding a protein required for complementation of a host cell auxotrophy, preferably a rich-media compatible auxotrophy.
  • the host cell is auxotrophic for thymidine ( ⁇ thyA), and the vector comprises the thymidylate synthase (thyA) gene.
  • the host cell is auxotrophic for diaminopimelic acid ( ⁇ dapA) 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.
  • the vector comprises a toxin gene.
  • the host cell comprises an anti-toxin gene encoding and/or required for the expression of an anti-toxin.
  • the toxin is Zeta and the anti-toxin is Epsilon.
  • the toxin is Kid, and the anti-toxin is Kis.
  • 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.
  • the gene encoding the toxin may be regulated by a moderate promoter.
  • 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)).
  • 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.
  • 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.
  • FIG.32 depicts a map of integration sites within the E. coli Nissle chromosome.
  • FIG.33 depicts three bacterial strains wherein the RFP gene has been successfully integrated into the bacterial chromosome at an integration site.
  • 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 propionate catabolism enzyme from the bacterial cytoplasm.
  • a native secretion mechanism e.g., gram positive bacteria
  • non-native secretion mechanism e.g., gram negative bacteria
  • 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.
  • secretion machineries may span one or both of the inner and outer membranes.
  • the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system.
  • Double membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance- nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference).
  • RTD resistance- nodulation-division
  • T7SS type VII secretion system
  • T2SS type VII secretion system
  • double membrane-spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell.
  • 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).
  • the genetically engineered bacteria of the invention further comprise a type III or a type III-like secretion system (T3SS) from Shigella,
  • 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.
  • the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the propionate catabolism enzyme from the bacterial cytoplasm.
  • the secreted molecule such as a heterologouse protein or peptide, e.g., a propionate catabolism enzyme, comprises a type III secretion sequence that allows the propionate catabolism enzyme to be secreted from the bacteria.
  • a flagellar type III secretion pathway is used to secrete the molecule of interest, e.g., a propionate catabolism enzyme.
  • 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.
  • 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.
  • a therapeutic peptide star
  • 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.
  • the secreted molecule such as a heterologouse protein or peptide, e.g., a propionate 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.
  • 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.
  • Figure 11 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.
  • 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.
  • 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.
  • 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.
  • Gram-positive bacteria e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii,
  • the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the propionate catabolism enzymefrom the bacterial cytoplasm.
  • 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.
  • One way to secrete properly folded proteins in gram-negative bacteria 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.
  • 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
  • Lpp is the most abundant polypeptide in the bacterial cell existing at ⁇ 500,000 copies per cell and functions as the primary‘staple’ of the bacterial cell wall to the peptidoglycan.1.
  • the engineered bacteria have one or more deleted or mutated membrane genes.
  • the engineered bacteria have a deleted or mutated lpp gene.
  • the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes.
  • the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes.
  • 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
  • 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.
  • 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, tolB, 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).
  • membrane or periplasmic protease genes e.g., selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl
  • 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.
  • 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.
  • the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.
  • Table 9 The tables below lists secretion systems for Gram positive bacteria and Gram negative bacteria.
  • one or more propionate catabolic enzymes described herein are secreted. In some embodiments, the one or more propionate catabolic enzymes described herein are further modified to improve secretion efficiency, decreased
  • PrpE is secreted, alone or in combination other propionate catabolic enzymes, e.g.,with one or more of accA1, pccB, mmcE, mutA, and mutB and/or one or more of prpB, prpC, prpD, and/or one or more of phaB, phaC, phaA.
  • one or more of accA1, pccB, mmcE, mutA, mutB are secreted.
  • one or more of prpB, prpC, prpD are secreted.
  • one or more of phaB, phaC, phaA are secreted.
  • any of the enzymes expressed by the genes described herein, e.g., in FIG.9, FIG.10, FIG.15, and FIG.20 may be combined.
  • the engineered bacteria may be evaluated in vivo, e.g., in an animal model.
  • Any suitable animal model of a disease or condition associated with catabolism of propionate may be used.
  • a hypomorphic mouse model of propionic acidemia as described by Guenzel et al. can be used (see, for example, Guenzel et al., 2013, Molecular. Ther., 21(7):1316-1323).
  • This PCCA-/- knock-out mouse lacks Pcca protein and accumulates high levels of propionylcarnitine and methyl citrate and dies within 36 hours of birth.
  • mouse model of methylmalonic acidemia has been generated by targeted deletion of a critical exon in the murine methylmalonyl-CoA mutase (Mut) gene (VENDITTI CP, et al/. Genetic and genomic systems to study methylmalonic acidemia (MMA) Mol Genet Metab.2005;84:207–208).
  • the Mut ⁇ / ⁇ mice display early neonatal lethality and faithfully replicate the severe phenotype of affected humans and display early neonatal lethality.
  • Studies in the Mut ⁇ / ⁇ mice have demonstrated progressive hepatic pathology and massive accumulation of methylmalonic acid in the liver near the time of death. This model has been extensively used to examine the effectiveness of rAAVs in the treatment of MMA.
  • the engineered bacterial cells may administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by measuring blood levels of
  • propionylcarnitine, acetylcarnitine, and/or methylcitrate before and after treatment see, for example, Guenzel et al., 2013.
  • the animal may be sacrificed, and tissue samples may be collected and analyzed.
  • a decrease in blood levels of propionylcarnitine, acetylcarnitine, and/or methylcitrate after treatment indicates that the engineered bacteria are effective for treating the disease.
  • ALE Adaptive laboratory evolution
  • 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.
  • a strain capable of high-affinity capture of said metabolite or biomolecule can be evolved via ALE.
  • 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.
  • 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.
  • a metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite.
  • 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.
  • 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.
  • the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques.
  • compositions comprising the genetically engineered bacteria described herein may be used to treat, manage, ameliorate, and/or prevent disorders associated with propionate 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.
  • the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic
  • 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 propionate catabolism gene(s) or gene cassette(s).
  • 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.
  • 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).
  • 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.
  • 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
  • the genetically engineered bacteria described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate-release, pulsatile-release, delayed- release, or sustained release).
  • Suitable dosage amounts for the genetically engineered bacteria may range from about 10 5 to 10 12 bacteria, e.g., approximately 10 5 bacteria, approximately 10 6 bacteria, approximately 10 7 bacteria, approximately 10 8 bacteria, approximately 10 9 bacteria, approximately 10 10 bacteria, approximately 10 11 bacteria, or approximately 10 11 bacteria.
  • the composition may be administered once or more daily, weekly, or monthly.
  • the composition may be administered before, during, or following a meal.
  • the pharmaceutical composition is administered before the subject eats a meal.
  • the pharmaceutical composition is administered currently with a meal.
  • the pharmaceutical composition is administered after the subject eats a meal.
  • 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.
  • 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.
  • 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.
  • viscous to semi- solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed.
  • Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts
  • 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.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • the pharmaceutical composition comprising the engineered bacteria may be formulated as a hygiene product.
  • 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.
  • 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.
  • Tablets or capsules can be prepared by conventional means with
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth
  • fillers e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate
  • lubricants e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica
  • disintegrants e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders
  • wetting agents e.g., sodium lauryl sulphate
  • the tablets may be coated by methods well known in the art.
  • a coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A- PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly
  • PEG/PD5/PDMS pentamethylcyclopentasiloxane/polydimethylsiloxane
  • PDMAAm poly N,N- dimethyl acrylamide
  • 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.
  • 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).
  • the pH profile may be modified.
  • 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.
  • 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.
  • Materials used for enteric coatings include Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacrylate), Cellulose acetate trimellitate (CAT), Poly(vinyl acetate phthalate) (PVAP) and Hydroxypropyl methylcellulose phthalate
  • CAP Cellulose acetate phthalate
  • CAT Cellulose acetate trimellitate
  • PVAP Poly(vinyl acetate phthalate)
  • HPMCP fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers.
  • Zein, Aqua-Zein an aqueous zein formulation containing no alcohol
  • amylose starch and starch derivatives e.g., amylose starch and starch derivatives
  • dextrins e.g., maltodextrin
  • enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylose acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate.
  • 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, EudragitTM S (poly(methacrylic acid, methyl methacrylate)1:2); Eudragit L100TM S (poly(methacrylic acid, methyl methacrylate)1:1); Eudragit L30DTM, (poly(methacrylic acid, ethyl acrylate)1:1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate)1:1) (EudragitTM L is an anionic polymer synthesized from methacrylic 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
  • Coating layers may also include polymers which contain
  • HPMC Hydroxypropylmethylcellulose
  • HPEC Hydroxypropylethylcellulose
  • HPC Hydroxypropylcellulose
  • HPEC hydroxypropylethylcellulose
  • HMPC hydroxymethylpropylcellulose
  • EHEC ethylhydroxyethylcellulose
  • HEMC hydroxyethylmethylcellulose
  • HMEC hydroxymethylethylcellulose
  • PHEC propylhydroxyethylcellulose
  • M H EC methylhydroxyethylcellulose
  • hydroxyethylcellulose CHEC
  • Methylcellulose 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, hydroxypropylcellulose phthalate (HPCP),
  • HPECP hydroxypropylethylcellulose phthalate
  • HPMCP hydroxyproplymethylcellulose phthalate
  • HPMCAS hydroxyproplymethylcellulose acetate succinate
  • Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non- aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol, or fraction
  • 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.
  • the genetically engineered bacteria of the disclosure may be formulated in a composition suitable for administration to pediatric subjects.
  • a 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.
  • a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.
  • 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.
  • the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life.
  • the gummy candy may also comprise sweeteners or flavors.
  • the composition suitable for administration to pediatric subjects may include a flavor.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition comprising the engineered bacteria may be a comestible product, for example, a food product.
  • 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.
  • the food product is a fermented food, such as a fermented dairy product.
  • the fermented dairy product is yogurt.
  • the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir.
  • the engineered bacteria are combined in a preparation containing other live bacterial cells intended to serve as probiotics.
  • the food product is a beverage.
  • the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts.
  • 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.
  • the pharmaceutical 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.
  • the pharmaceutical 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 express
  • composition is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.
  • 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.
  • 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,
  • Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges e.g., of gelatin
  • suitable powder base such as lactose or starch.
  • 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.
  • 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).
  • 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.
  • 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.
  • a single dosage form may be administered over a period of time, e.g., by infusion.
  • 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.
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release.
  • 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).
  • 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.
  • 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.
  • 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, LD 50 , ED 50 , EC 50 , and IC 50 may be determined, and the dose ratio between toxic and therapeutic effects (LD 50 /ED 50 ) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.
  • 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.
  • the pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • 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.
  • 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.
  • 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.
  • Another aspect of the disclosure provides methods of treating a disease associated with catabolism of propionate in a subject, or symptom(s) associated with the disease associated with the catabolism of propionate in a subject.
  • the disorder involving the catabolism of propionate is a metabolic disorder involving the abnormal catabolism of propionate.
  • Metabolic diseases associated with abnormal catabolism of propionate include propionic acidemia (PA) and methylmalonic acidemia (MMA), as well as severe nutritional vitamin B 12 deficiencies.
  • the disease associated with abnormal catabolism of propionate is propionic acidemia.
  • the disease associated with abnormal catabolism of propionate is methylmalonic acidemia.
  • the disease associated with abnormal catabolism of propionate is a vitamin B 12 deficiency.
  • the disease is propionic acidemia.
  • Propionic acidemia also known as propionyl-CoA carboxylase deficiency, PROP, PCC deficiency, ketotic hyperglycinemia, ketotic glycinemia, and hyper glycinemia with ketoacidosis and leukopenia
  • PROP propionyl-CoA carboxylase deficiency
  • PCC autosomal recessive disorder caused by impaired activity of Propionyl CoA carboxylase (PCC; EC 6.4.1.3).
  • PCC is responsible for converting propionyl CoA into methylmalonyl CoA.
  • Propionyl CoA Carboxylase is a dodecameric enzyme comprised of alpha and beta subunits.
  • the alpha subunit of PCC also called PCCA; NM_000282
  • the beta subunit also called PCCB; NM_000532
  • Propionic Acidemia (Perez et al., Mol. Genet Metabol., 78(1):59-67, 2003), including missense mutations, nonsense mutations, point exonic mutations affecting splicing, splicing mutations, insertions and deletions.
  • the disease is methylmalonic acidemia.
  • Methylmalonic acidemia also known as methylmalonic aciduria or isolated methylmalonic acidemia, is an autosomal recessive disorder caused by impaired activity of one of several genes: MUT (OMIM 251000), MMAA (OMIM 251100), MMAB (OMIM 251110), MMACHC (OMIM 27740), MMADHC (OMIM 277410), or LMBRD1 (OMIM 277380).
  • MUT OMIM 251000
  • MMAA OMIM 251100
  • MMAB OMIM 251110
  • MMACHC OMIM 27740
  • MMADHC OMIM 277410
  • LMBRD1 OMIM 277380
  • MUT is responsible for converting methylmalonyl CoA into succinyl CoA and requires a vitamin B 12 -derived prosthetic group, adenosyl fimin (also known as AdoCbl) to function.
  • AdoCbl adenosyl enhanced glutathione
  • the methylmalonic aciduria type A protein, mitochondrial also known as MMAA aides AdoCbl loading onto MUT.
  • Cob(l)yrinic acid, a,c-diamind adenosyltransferase, mitochondrial is an enzyme that catalyzes the final step in the conversion of vitamin B 12 into adenosylcobalamin (AdoCbl).
  • MMA patients with MMA are unable to properly process methylmalonyl CoA, which can lead to the toxic accumulation of methylmalonyl CoA and methylmalonic acid in the blood, cerebrospinal fluid and tissues.
  • Clinical manifestations of the disease vary depending on the degree of enzyme deficiency and include seizures, vomiting, lethargy, hypotonia, encephalopathy, developmental delay, failure to thrive, and secondary
  • hyperammonemia (Deodato et al., Methylmalonic and propionic aciduria, Am. J. Med. Genet. C. Semin. Med. Genet, 142(2):104-112, 2006).
  • Detectable urinary organic acids useful for diagnosis and markers include, but are not limited to, N-propionylglycine, N-tiglyglycine, 2-methyl-3-oxovaleric acid, 3- hydroxy-2-methylbutyric acid, 2 methyl-3-oxobutyric acid, 3-hydroxy-n-valeric acid, 3-oxo- n-valeric acid
  • compositions comprising the engineered bacterial cells may be used to treat metabolic diseases involving the abnormal catabolism of propionate, such as PA and MMA.
  • the subject having PA has a mutation in a PCCA gene. In another embodiment, the subject having PA has a mutation in the PCCB gene.
  • the subject having MMA has a mutation in the MUT gene. In another embodiment, the subject having MMA has a mutation in the MMAA gene. In another embodiment, the subject having MMA has a mutation in the MMAB gene. In another embodiment, the subject having MMA has a mutation in the MMACHC gene. In another embodiment, the subject having MMA has a mutation in the MMADHC gene. In another embodiment, the subject having MMA has a mutation in the LMBRD1 gene.
  • the disclosure provides methods for decreasing the plasma level of propionate, propionyl CoA, and/or methylmalonic CoA in a subject by administering a pharmaceutical composition comprising a bacterial cell to the subject, thereby decreasing the plasma level of the propionate, propionyl CoA, and/or methylmalonic CoA in the subject.
  • the subject has a disease or disorder involving the catabolism of propionate.
  • the disorder involving the catabolism of propionate is a metabolic disorder involving the abnormal catabolism of propionate.
  • the disorder involving the catabolism of propionate is propionic acidemia.
  • the disorder involving the catabolism of propionate is methylmalonic acidemia.
  • the disorder involving the catabolism of propionate is a vitamin B 12 deficiency.
  • the disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to seizures, vomiting, lethargy, hypotonia, encephalopathy, developmental delay, failure to thrive, liver failure, and/or secondary hyperammonemia.
  • the disease is secondary to other conditions, e.g., liver disease.
  • the bacterial cells are capable of catabolizing propionate, propionyl CoA and/or methylmalonyl CoA in a subject in order to treat a disease associated with catabolism of propionate.
  • the bacterial cells are delivered simultaneously with dietary protein.
  • the bacterial cells are delivered simultaneously with L-carnitine.
  • the bacterial cells and dietary protein are delivered after a period of fasting or protein-restricted dieting.
  • a patient suffering from a disorder involving the catabolism of propionate e.g., PA or MMA, may be able to resume a substantially normal diet, or a diet that is less restrictive than a protein-free or very low-protein diet.
  • the bacterial cells may be capable of catabolizing propionate, propionyl CoA, and/or methylmalonyl CoA from additional sources, e.g., the blood, in order to treat a disease associated with the catabolism of propionate.
  • the bacterial cells need not be delivered simultaneously with dietary protein, and a gradient is generated, e.g., from blood to gut, and the engineered bacteria catabolize the propionate, propionyl CoA, and/or methylmalonyl CoA and reduce plasma levels of the propionate, propionyl CoA, and/or methylmalonyl CoA.
  • 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.
  • the genetically engineered bacteria disclosed herein are administered orally, e.g., in a liquid suspension.
  • the genetically engineered bacteria are lyophilized in a gel cap and administered orally.
  • the genetically engineered bacteria 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.
  • the pharmaceutical composition described herein is administered to reduce propionate, propionyl CoA, and/or methylmalonyl CoA levels in a subject.
  • the methods of the present disclosure reduce the propionate, propionyl CoA, and/or methylmalonyl CoA levels in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more.
  • the methods of the present disclosure reduce the propionate, propionyl CoA, and/or methylmalonyl CoA 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 some embodiments, reduction is measured by comparing the propionate, propionyl CoA, and/or methylmalonyl CoA level in a subject before and after administration of the pharmaceutical composition. In one embodiment, the propionate, propionyl CoA, and/or methylmalonyl CoA level is reduced in the gut of the subject.
  • the propionate, propionyl CoA, and/or methylmalonyl CoA level is reduced in the blood of the subject. In another embodiment, the propionate, propionyl CoA, and/or methylmalonyl CoA level is reduced in the plasma of the subject. In another embodiment, the propionate, propionyl CoA, and/or methylmalonyl CoA level is reduced in the brain of the subject.
  • the pharmaceutical composition described herein is administered to reduce propionate, propionyl CoA, and/or methylmalonyl CoA levels in a subject to normal levels. In another embodiment, the pharmaceutical composition described herein is administered to reduce propionate, propionyl CoA, and/or methylmalonyl CoA levels in a subject to below a normal level.
  • the method of treating the disorder involving the catabolism of propionate 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.
  • the method of treating the disorder involving the catabolism of propionate, e.g., PA or MMA allows one or more symptoms of the condition or disorder to improve by at least about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold.
  • propionate, propionyl CoA, and/or methylmalonyl CoA 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, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal.
  • the methods may include administration of the compositions of the disclosure to reduce levels of the propionate, propionyl CoA, and/or methylmalonyl CoA.
  • the methods may include administration of the compositions of the disclosure to reduce the propionate, propionyl CoA, and/or methylmalonyl CoA to undetectable levels in a subject. In some embodiments, the methods may include administration of the compositions of the disclosure to reduce the propionate, propionyl CoA, and/or methylmalonyl CoA 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 propionate, propionyl CoA, and/or methylmalonyl CoA levels prior to treatment.
  • the engineered bacterial cells produce a propionate catabolism enzyme under exogenous environmental conditions, such as the low-oxygen environment of the mammalian gut, to reduce levels of propionate, propionyl CoA, and/or methylmalonyl CoA in the 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.
  • exogenous environmental conditions such as the low-oxygen environment of the mammalian gut
  • Propionate, propionyl CoA, and/or methylmalonyl CoA levels may be measured by methods known in the art, e.g., blood sampling and mass spectrometry as described in Guenzel et al., 2013, Molecular Ther., 21(7):1316-1323.
  • propionate catabolism enzyme e.g., PrpBCDE
  • expression is measured by methods known in the art.
  • propionate catabolism enzyme activity is measured by methods known in the art to assess PrpBCDE activity (see propionate catabolism enzyme sections, supra).
  • propionate catabolism enzyme activity is measured by methods known in the art to assess activity of a PHA pathway circuit described herein.
  • propionate catabolism enzyme activity is measured by methods known in the art to assess the activity of a MMCA circuit described herein.
  • the genetically engineered bacteria is E. coli Nissle.
  • the genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009), or by activation of a kill switch, several hours or days after administration.
  • the pharmaceutical composition comprising the engineered bacteria may be re-administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice can be determined.
  • the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.
  • the bacterial cells are administered to a subject once daily.
  • 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.
  • 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.
  • the methods disclosed herein may comprise administration of a composition alone or in combination with one or more additional therapies, e.g., phenylbutyrate, thiamine supplementation, L-carnitine, and/or a low-protein diet.
  • additional therapies e.g., phenylbutyrate, thiamine supplementation, L-carnitine, and/or a low-protein diet.
  • the pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents.
  • agent(s) should be compatible with the bacteria, e.g., the agent(s) must not interfere with or kill the bacteria.
  • the agent(s) should be compatible with the bacteria, e.g., the agent(s) must not interfere with or kill the bacteria.
  • the pharmaceutical composition is administered with food.
  • the pharmaceutical composition is administered before or after eating food.
  • the pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g., low-protein diet and amino acid supplementation.
  • the dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder.
  • the appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.
  • the methods may further comprise isolating a plasma sample from the subject prior to administration of a composition and determining the level of the propionate, propionyl CoA and/or methylmalonyl CoA in the sample. In some embodiments, the methods may further comprise isolating a plasma sample from the subject after to
  • composition administration of a composition and determining the level of the propionate, propionyl CoA and/or methylmalonyl CoA in the sample.
  • the methods further comprise comparing the level of the propionate, propionyl CoA, and/or methylmalonyl CoA in the plasma sample from the subject after administration of a composition to the subject to the plasma sample from the subject before administration of a composition to the subject.
  • a reduced level of the propionate, propionyl CoA, and/or methylmalonyl CoA in the plasma sample from the subject after administration of a composition indicates that the plasma levels of the propionate, propionyl CoA, and/or methylmalonyl CoA are decreased, thereby treating the disorder involving the catabolism of propionate in the subject.
  • the plasma level of the propionate, propionyl CoA, and/or methylmalonyl CoA 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 plasma level in the sample before administration of the pharmaceutical composition.
  • the plasma level of the propionate, propionyl CoA, and/or methylmalonyl CoA 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 plasma level in the sample before
  • the methods further comprise comparing the level of the propionate, propionyl CoA, and/or methylmalonyl CoA in the plasma sample from the subject after administration of a composition to a control level of propionate, propionyl CoA, and/or methylmalonyl CoA.
  • the methods may further comprise isolating a plasma sample from the subject prior to administration of a composition and determining the level of the propionate, propionyl CoA and/or methylmalonyl CoA in the sample.
  • the methods may further comprise isolating a plasma sample from the subject after to
  • composition administration of a composition and determining the level of the propionate, propionyl CoA and/or methylmalonyl CoA in the sample.
  • the methods further comprise comparing the level of methylcitrate, propionylcarnitine, and/or acetylcarnitine, and/or the propionylcarnitine to acetylcarnitine ratio in the plasma sample from the subject after administration of a composition to the subject to the plasma sample from the subject before administration of a composition to the subject.
  • a reduced level of methylcitrate, propionylcarnitine, and/or acetylcarnitine, and/or the propionylcarnitine to acetylcarnitine ratio in the plasma sample from the subject after administration of a composition to the subject to the plasma sample from the subject before administration of a composition to the subject.
  • a reduced level of methylcitrate is one embodiment.
  • propionylcarnitine, and/or acetylcarnitine the propionylcarnitine to acetylcarnitine ratio in the plasma sample from the subject after administration of a composition indicates that the plasma levels of methylcitrate, propionylcarnitine, and/or acetylcarnitine are decreased, thereby treating the disorder involving the catabolism of propionate in the subject.
  • the plasma level of methylcitrate, propionylcarnitine, and/or acetylcarnitine, and/or the propionylcarnitine to acetylcarnitine ratio 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 plasma level in the sample before
  • the plasma level of methylcitrate, propionylcarnitine, and/or acetylcarnitine, and/or the propionylcarnitine to acetylcarnitine ratio 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 plasma level in the sample before administration of the pharmaceutical composition.
  • the methods further comprise comparing the level of methylcitrate, propionylcarnitine, and/or acetylcarnitine, and/or the propionylcarnitine to acetylcarnitine ratio in the plasma sample from the subject after administration of a composition to a control level of methylcitrate, propionylcarnitine, and/or acetylcarnitine.

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