AU2016303662A1

AU2016303662A1 - Bacteria engineered to treat disorders involving propionate catabolism

Info

Publication number: AU2016303662A1
Application number: AU2016303662A
Authority: AU
Inventors: Dean Falb; Vincent ISABELLA; Jonathan Kotula; Paul Miller; Yves Millet; Alex TUCKER
Original assignee: Synlogic Operating Co Inc
Current assignee: Synlogic Operating Co Inc
Priority date: 2015-07-31
Filing date: 2016-07-29
Publication date: 2018-02-22
Also published as: CA2996535A1; JP2018521674A; WO2017023818A1; EP3328988A1

Abstract

The present disclosure provides engineered bacterial cells comprising a heterologous gene encoding a propionate catabolism enzyme. In another aspect, the engineered bacterial cells further comprise at least one heterologous gene encoding a transporter of propionate or a kill switch. The disclosure further provides pharmaceutical compositions comprising the engineered bacteria, and methods for treating disorders involving the catabolism of propionate, such as Propionic Acidemia and Methylmalonic Acidemia, using the pharmaceutical compositions.

Description

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 threedimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term “peptide” or “polypeptide” may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.

[0104] An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required.

Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant

DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms “fragment,” “variant,” “derivative” and “analog” include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or

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PCT/US2016/044922 more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be nonnaturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or nonconservative amino acid substitutions, deletions or additions.

[0105] Polypeptides also include fusion proteins. As used herein, the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term “fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins.“Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. “Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. 0., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, He, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

[0106] As used herein, the term “sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at

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PCT/US2016/044922 least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones.

[0107] As used herein the term “linker”, “linker peptide” or “peptide linkers” or “linker” refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains. As used herein the term “synthetic” refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.

[0108] As used herein the term “codon-optimized” refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A “codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the -39WO 2017/023818

PCT/US2016/044922 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.

[0109] As used herein, the terms “secretion system” or “secretion protein” refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule,

e.g., polypeptide from the microbial, e.g., bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex

e.g.,HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type

V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the polypeptide to be secreted include a “secretion tag” of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the

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

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PCT/US2016/044922 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.

[0110] As used herein a pharmaceutical composition refers to a preparation of bacterial cells with other components such as a physiologically suitable carrier and/or excipient.

[0111] The phrases physiologically acceptable carrier and pharmaceutically acceptable carrier which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.

[0112] The term excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[0113] The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a disease. 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. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

[0114] As used herein, the term bacteriostatic” or cytostatic” refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of engineered bacterial cell of the disclosure.

[0115] As used herein, the term “bactericidal” refers to a molecule or protein which is capable of killing the engineered bacterial cell of the disclosure.

[0116] As used herein, the term “toxin” refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the engineered bacterial cell of the disclosure, or which is capable of killing the engineered bacterial cell of the disclosure. The term “toxin” is intended to include bacteriostatic proteins and bactericidal proteins. The term “toxin” is intended to include, but not limited to, lytic proteins, bacteriocins (e.g., microcins -41WO 2017/023818

PCT/US2016/044922 and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term “anti-toxin” or “antitoxin,” as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term antitoxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.

[0117] The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

[0118] The phrase “and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may be used interchangeably with “at least one of’ or “one or more of’ the elements in a list.

[0119] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Bacterial Strains [0120] The disclosure provides a bacterial cell that comprises at least one heterologous gene encoding a propionate catabolism enzyme. In some embodiments, the bacterial cell is a non-pathogenic bacterial cell. In some embodiments, the bacterial cell is a commensal bacterial cell. In some embodiments, the bacterial cell is a probiotic bacterial cell.

[0121] In certain embodiments, the bacterial cell is selected from the group consisting of a Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Clostridium scindens, Escherichia coli, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, and Oxalobacter formigenes bacterial cell. In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron

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PCT/US2016/044922 bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium animalis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is a Clostridium scindens bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell.

In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Oxalobacter formigenes bacterial cell. In another embodiment, the bacterial cell does not include Oxalobacter formigenes.

[0122] In one embodiment, the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.

[0123] In some embodiments, the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et 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 α-hemolysin, Pfimbrial adhesins) (Schultz, 2008), and E. coli Nissle does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic (Sonnenbom et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. It is commonly accepted that E. coli Nissle’s therapeutic efficacy and safety have convincingly been proven (Ukena et al., 2007).

[0124] In one embodiment, the engineered bacterial cell does not colonize the subject.

[0125] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. Furthermore, genes from one or more different species can be introduced into one another, e.g., a gene from Lactobacillus plantarum or Methanobrevibacter smithii 3142 can be expressed in Escherichia coli.

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PCT/US2016/044922 [0126] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is an engineered bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells.

[0127] In another aspect, the disclosure provides an engineered bacterial culture which comprises engineered bacterial cells.

[0128] In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette(s) are present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette(s) is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[0129] In some embodiments, the genetically engineered bacteria is an auxotroph or a conditional auxotroph. In one embodiment, the genetically engineered bacteria is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[0130] In some embodiments, the genetically engineered bacteria further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of an promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD· In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

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PCT/US2016/044922 [0131] In some embodiments, the genetically engineered bacteria is an auxotroph and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.

[0132] In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette(s) are present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette(s) are present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[0133] In one aspect, 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. In another embodiment, 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. In one embodiment, 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.

[0134] 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). Non-limiting examples of such enzymes and propionate metabolic pathways are described herein. For example, 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. In some aspects, the disclosure provides a bacterial cell that comprises one or more heterologous gene sequence(s) and/or gene cassette(s) encoding one or more 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.

[0135] In certain embodiments, the genetically engineered bacteria are obligate anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are facultative anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are aerobic bacteria. In some embodiments, the genetically engineered bacteria are Grampositive bacteria. In some embodiments, the genetically engineered bacteria are Gram-45WO 2017/023818

PCT/US2016/044922 positive bacteria and lack LPS. In some embodiments, the genetically engineered bacteria are Gram-negative bacteria. In some embodiments, the genetically engineered bacteria are

Gram-positive and obligate anaerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and facultative anaerobic bacteria. In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter,

Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria,

Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio,

Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum,

Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55,

Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT, Clostridium paraputrificum, Clostridium pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum,

Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum,

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

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

Clostridium butyricum, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus,

Lactobacillus plantarum, Lactobacillus reuteri, and Eactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the -46WO 2017/023818

PCT/US2016/044922 bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell.

[0136] In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et al., 2007).

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

[0137] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria.

Furthermore, genes from one or more different species can be introduced into one another,

e.g., 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 freudenreichii, or matB, derived from Rhodopseudomonas palustris, can be expressed in Escherichia coli. In some embodiments, the genes are codon optimized, e.g., for expression in E. coli. In one -47WO 2017/023818

PCT/US2016/044922 embodiment, the recombinant bacterial cell does not colonize the subject having the disorder. Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009). In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention.

[0138] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells disclosed herein.

[0139] In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein. In one aspect, the disclosure provides a recombinant bacterial culture which reduces levels of propionate in the media of the culture. In one embodiment, 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. In another embodiment, 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. In one embodiment, 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.

[0140] In some embodiments of the above described genetically engineered bacteria, 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. In other embodiments, 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 lowoxygen or anaerobic conditions, such as any of the promoters disclosed herein. In some embodiments of the above described genetically engineered bacteria, the gene encoding a 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. In other embodiments, 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.

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PCT/US2016/044922 [0141] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding a propionate catabolism enzyme is an auxotroph. In one embodiment, the genetically engineered bacteria is an auxotroph selected from a cysE, glnA, ilvD, leuB, lys A, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph. ,In some embodiments, 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.

[0142] In some embodiments, 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. In certain embodiments, the propionate transporter is MctC, PutP_6, or any other propionate transporters described herein. In certain embodiments, the bacterial cell contains gene sequence encoding MctC, PutP_6, or any other propionate transporters described herein.

[0143] In some embodiments, 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.

[0144] In some embodiments, 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.

[0145] In some embodiments, the genetically engineered bacteria comprising a propionate catabolism enzyme further comprise a kill-switch circuit, such as any of the killswitch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence.

In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an

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PCT/US2016/044922 enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

[0146] In some embodiments, 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.

[0147] In some embodiments of the above described genetically engineered bacteria, 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. 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 in the bacterial chromosome. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present on a plasmid in the bacterium. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome.

Inducible Promoters [0148] In some embodiments, 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. In some embodiments, bacterial cell comprises two or more distinct propionate catabolism

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PCT/US2016/044922 enzymes. In some embodiments, the genetically engineered bacteria comprise multiple copies of the same propionate catabolism enzyme gene. In some embodiments, the genetically engineered bacteria comprise multiple copies of different propionate catabolism enzyme genes. In some embodiments, 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.

[0149] In some embodiments, 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. In some embodiments, bacterial cell comprises two or more distinct copies of the at least one gene encoding a propionate transporter. In some embodiments, the genetically engineered bacteria comprise multiple copies of the same at least one gene encoding a propionate transporter. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

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PCT/US2016/044922 [0150] 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 transporter, is directly induced by exogenous environmental conditions. 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 transporter, is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by 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 coadministered with the bacterial cell.

[0151] In some embodiments, 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. In some embodiments, bacterial cell comprises two or more distinct copies of the at least one gene encoding a propionate binding protein. In some embodiments, the genetically engineered bacteria comprise multiple copies of the same at least one gene encoding a propionate binding protein. In some embodiments, 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. In some embodiments, 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.

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PCT/US2016/044922 [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 environmental conditions. 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 indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by 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.

FNR dependent regulation [0153] In certain embodiments, 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. In E. coli, 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.

[0154] 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.

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Table 2 FNR responsive promoters

FNR Responsive Promoter	Sequence
SEQ ID NO: 1	GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTATCGTCGTCCGGCCT TTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACA AACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTA AG G AGTATATAAAG GTG AATTTG ATTTACATCAATA AG CG G G GTTG CTG A ATCGTTAAG GTAG G CGGTAATAGAAAAGAAATCGAGGCAAAA
SEQ ID NO: 2	ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTCATGCATGCATCAAA AAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCG TTACGTGGGCTTCGACTGTAAATCAGAAAGGAGAAAACACCT
SEQ ID NO: 3	GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTATCGTCGTCCGGCCT TTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACA AACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTA AG G AGTATATAAAG GTG AATTTG ATTTACATCAATA AG CG G G GTTG CTG A ATCGTTAAGG ATCC CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT
SEQ ID NO: 4	CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTCATGCATGCATCAA AAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCC GGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT
SEQ ID NO: 5	AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTAACAAAAGCAAT TTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCA TTCAGGGCAATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA CAT
SEQ ID NO: 6	ATCCCCATCACTCTTGATGGAGATCAATTCCCCAAGCTGCTAGAGCGTTACCTTGCCCTTAAACAT TAGCAATGTCGATTTATCAGAGGGCCGACAGGCTCCCACAGGAGAAAACCG
SEQ ID NO: 7	CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACCTTGCCCTTAAACATTAGCAATGT CGATTTATCAGAGGGCCGACAGGCTCCCACAGGAGAAAACCG

Table 3. FNR Promoter Sequences

SEQ ID NO	FNR-responsive regulatory region Sequence
	GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACT
nirBl	ATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCT ATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGAC
SEQ ID NO: 8	AATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAG GAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAAT CGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA

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nirB2 SEQ ID NO: 9	CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTACAGCAA ACGGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTC AGCCGTCACCGTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCC GGACGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGC ATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGA AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATAT ACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGG GTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA atgtttgtttaactttaagaaggagatatacat
nirB3 SEQ ID NO: 10	GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCACT ATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCT ATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAATATCAGAC AATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCATTAAG GAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAAT CGTTAAGGTAGGCGGTAATAGAAAAGAAATCGAGGCAAAA
yri/Z SEQ ID NO: 11	ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGC TCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATT TCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCGACTGT AAATCAGAAAGGAGAAAACACCT
nirB+RBS SEQ ID NO: 12	GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACT ATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCT ATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGAC AATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAG GAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAAT CGTTAAGGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATA TACAT
ydfZ+RBS SEQ ID NO: 13	CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGG CTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATAT TTCACTCGACAGGAGTATTTATATTGCGCCCGGATCCCTCTAGAAATAAT TTTGTTTAACTTTAAGAAGGAGATATACAT
fnrSl SEQ ID NO: 14	AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGT TGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAG TTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTT GGATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

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fnrS2 SEQ ID NO: 15	AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGT TGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAG TTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTT GGATCCAAAGTGAACTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
TATACAT
nirB+crp SEQ ID NO: 16	TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGTCAG CATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCACTATCGT CGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCTATAAA CCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAATATCAGACAATTC CGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCATTAAGGAGTA TATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTA AGGTAGaaatgtgatctagttcacatttGCGGTAATAGAAAAGAAATCGA GGCAAAAa t gtttgtttaactttaagaaggagatatacat
fnrS+crp SEQ ID NO: 17	AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGT TGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAG TTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCaa atgtgatctagttcacattttttgtttaactttaagaaggagatatacat

[0155] In one embodiment, 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. In yet another embodiment, 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.

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PCT/US2016/044922 [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. In other embodiments, 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. Accordingly, in some embodiments, the FNR responsive promoter has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding any of SEQ ID NOs: 1-43.

[0157] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a 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). In alternate embodiments, 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). In alternate embodiments, 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). In these embodiments, catabolism of propionate and/or its metabolites is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.

[0158] In some embodiments, 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,

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PCT/US2016/044922 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. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.

[0159] In some embodiments, the genetically engineered bacteria comprise a wildtype 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. In some embodiments, the genetically engineered bacteria comprise a wildtype oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the 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 transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., 2006).

[0160] In some embodiments, the bacterial cells disclosed herein comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator,

e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the propionate catabolism enzyme are present on different plasmids. In some embodiments, the gene -58WO 2017/023818

PCT/US2016/044922 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.

[0161] In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the 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. 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 propionate transporter and/or the at least one gene encoding a propionate binding protein are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the propionate catabolism enzyme and/or the transporter of propionate and /or metabolites thereof and/or the propionate binding protein. In some embodiments, 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. In some embodiments, the transcriptional regulator and the propionate catabolism enzyme are divergently transcribed from a promoter region.

RNS dependent regulation [0162] In some embodiments, the genetically engineered bacteria comprise a gene encoding a propionate catabolism enzyme that is expressed under the control of an inducible promoter. In some embodiments, 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

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PCT/US2016/044922 inflammatory conditions. In one embodiment, 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.

[0163] As used herein, “reactive nitrogen species” and “RNS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress.

RNS includes, but is not limited to, nitric oxide (NO·), peroxynitrite or peroxynitrite anion (ONOO-), nitrogen dioxide (·ΝΟ2), dinitrogen trioxide (N2O3), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOCO2-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

[0164] As used herein, “RNS-inducible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g., a propionate catabolism enzyme gene sequence(s), e.g., any of the amino acid catabolism enzymes described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

[0165] As used herein, “RNS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene -60WO 2017/023818

PCT/US2016/044922 expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., propionate catabolism enzyme gene sequence(s), propionate transporter sequence(s), propionate binding protein(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

[0166] As used herein, “RNS-repressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNSrepressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

[0167] As used herein, a “RNS-responsive regulatory region” refers to a RNSinducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 4.

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

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

[0168] In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of an 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. For example, the tunable regulatory region is a RNS-inducible regulatory region, and 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; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene or genes. Subsequently, when 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.

[0169] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

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PCT/US2016/044922 [0170] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR “is an NOresponsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide” (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNSresponsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.

[0171] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) “promotes the expression of the nir, the nor and the nos genes” in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more amino acid catabolism enzymes. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

[0172] In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0173] In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is “an Rrf2-63WO 2017/023818

PCT/US2016/044922 type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism” (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a propionate catabolism enzyme gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene or genes and producing the encoding an amino acid catabolism enzyme(s).

[0174] In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[0175] In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0176] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express an amino acid catabolism

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PCT/US2016/044922 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. In one aspect of these embodiments, the RNSsensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, Cl, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene or genes is expressed.

[0177] A RNS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNSsensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012).

[0178] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter.

In some instances, it may be advantageous to express the RNS-sensing transcription factor

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PCT/US2016/044922 under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0179] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0180] In some embodiments, the genetically engineered bacteria comprise a RNSsensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity.

[0181] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the

RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the

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

RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for -66WO 2017/023818

PCT/US2016/044922 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.

[0182] In some embodiments, the genetically engineered bacteria comprise a wildtype 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. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS-responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the propionate catabolism enzyme in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein in the presence of RNS.

[0183] In some embodiments, the gene or gene cassette for producing the antiinflammation and/or gut barrier function enhancer molecule is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0184] In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of a 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. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

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ROS-dependent regulation [0185] In some embodiments, 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. In some embodiments, 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. In one embodiment, the gene for producing the propionate catabolism enzyme is expressed under the control of a cellular damageddependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.

[0186] As used herein, “reactive oxygen species” and “ROS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metalcatalyzed 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 (·ΟΗ), superoxide or superoxide anion (·Ο2-), singlet oxygen (102), ozone (03), carbonate radical, peroxide or peroxyl radical (·Ο2-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOCl), nitric oxide (NO·), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al., 2014).

[0187] As used herein, “ROS-inducible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene

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PCT/US2016/044922 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). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

[0188] As used herein, “ROS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more amino acid catabolism enzyme(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

[0189] As used herein, “ROS-repressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROSrepressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

[0190] As used herein, a “ROS-responsive regulatory region” refers to a ROSinducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible

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PCT/US2016/044922 regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 5.

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

ROS-sensing transcription factor:	Primarily capable of sensing:	Examples of responsive genes, promoters, and/or regulatory regions:
OxyR	H₂O₂	ahpC; ahpF; dps; dsbG; fhuF; flu; fur; gor; grxA; hemH; katG; oxyS; sufA; sufB; sufC; sufD; sufE; sufS; trxC; uxuA; yaaA; yaeH; yaiA; ybjM; ydcH; ydeN; ygaQ; yljA; ytfK
PerR	h₂o₂	katA; ahpCF; mrgA; zoaA; fur; hemAXCDBF; srfA
OhrR	Organic peroxides NaOCl	ohrA
SoxR	•O₂' NO· (also capable of sensing H₂O₂)	soxS
RosR	H₂O₂	rbtT; tnp!6a; rluCl; tnp5a; mscF; tnp2d; phoD; tnpl5b; pstA; tnp5b; xylC; gabDl; rluC2; cgtS9; azlC; narKGHJI; rosR

[0191] In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of an amino acid catabolism enzyme, thus controlling expression of the propionate catabolism enzyme relative to ROS levels. For example, 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

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PCT/US2016/044922 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.

[0192] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0193] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR “functions primarily as a global regulator of the peroxide stress response” and is capable of regulating dozens of genes, e.g., “genes involved in H2O2 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)” and “OxyS, a small regulatory RNA” (Dubbs et al., 2012). The genetically engineered bacteria may comprise any suitable ROS-responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene. In the presence of ROS, e.g., H2O2, 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. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROSinducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.

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PCT/US2016/044922 [0194] In alternate embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is “activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression” (Koo et al., 2003). “SoxR is known to respond primarily to superoxide and nitric oxide” (Koo et al., 2003), and is also capable of responding to H2O2. The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al., 2003; Table

1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., an amino acid catabolism enzyme. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked 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.

[0195] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0196] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR “binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event,” but oxidized OhrR is “unable to bind its DNA target” (Duarte et al., 2010). OhrR is a “transcriptional repressor [that]... senses both organic peroxides and NaOCl” (Dubbs et al., 2012) and is “weakly activated by 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). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene. In the presence of ROS, e.g., NaOCl, an OhrR transcription factor senses ROS and no longer binds

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PCT/US2016/044922 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.

[0197] OhrR is a member of the MarR family of ROS-responsive regulators. “Most members of the MarR family are transcriptional repressors and often bind to the -10 or -35 region in the promoter causing a steric inhibition of RNA polymerase binding” (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012).

[0198] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is “a MarR-type transcriptional regulator” that binds to an “18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA” and is “reversibly inhibited by the oxidant 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 (cgl322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cgl426), two putative FMN reductases (cgll50 and cgl850), and four putative monooxygenases (cg0823, cgl848, cg2329, and cg3084)” (Bussmann et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., an amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein. In the presence of ROS, e.g., H2O2, 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

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PCT/US2016/044922 binding protein gene and producing the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein.

[0199] In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[0200] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0201] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR “when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)” (Marinho et al., 2014). PerR is a “global regulator that responds primarily to 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

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PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g.,

Dubbs et al., 2012; Table 1).

[0202] In these embodiments, 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. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, Cl, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., an amino acid catabolism enzyme. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., 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.

[0203] A ROS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although “OxyR is primarily thought of as a transcriptional activator under oxidizing conditions... OxyR can function as either a repressor or activator under both oxidizing and reducing conditions” (Dubbs et al., 2012), and OxyR “has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)” (Zheng et al., 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et 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. In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that -75WO 2017/023818

PCT/US2016/044922 is activated by RosR. In addition, “PerR-mediated positive regulation has also been observed.. .and appears to involve PerR binding to distant upstream sites” (Dubbs et al.,

2012). In some embodiments, the genetically engineered bacteria comprise any suitable

ROS-responsive regulatory region from a gene that is activated by PerR.

[0204] One or more types of ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, “OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.

[0205] Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 6. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 18, 19, 20, or 21, or a functional fragment thereof.

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

Regulatory sequence

01234567890123456789012345678901234567890123456789

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Regulatory sequence	01234567890123456789012345678901234567890123456789
katG (SEQ ID NO: 18)	TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACA GAGCACAAAATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGT TATCAGCCTTGTTTTCTCCCTCATTACTTGAAGGATATGAAGCTA AAACCCTTTTTTATAAAGCATTTGTCCGAATTCGGACATAATCA AAAAAGCTTAATTAAGATCAATTTGATCTACATCTCTTTAACCA ACAATATGTAAGATCTCAACTATCGCATCCGTGGATTAATTC AATTATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACT GTAGAGGGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAA GGTACC
dps (SEQ ID NO: 19)	TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACG CTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCG
GAACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGA CATAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGA GAAAGGTACC
ahpC (SEQ ID NO: 20)	GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC AAAGTTCACAAAGTTGTCTTACGAAGGTTGTAAGGTAAAACTT ATCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAAAT TGGTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATG CGAATTCATTAAAGAGGAGAAAGGTACC
oxyS (SEQ ID NO: 21)	CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGC GATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTC
TGACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGT ACC

[0206] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

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PCT/US2016/044922 [0207] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0208] In some embodiments, the genetically engineered bacteria comprise a ROSsensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROSsensing transcription factor, e.g., OxyR, is deleted or mutated to reduce or eliminate wildtype activity.

[0209] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROSsensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

[0210] In some embodiments, the genetically engineered bacteria comprise a wildtype gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a corresponding regulatory region, e.g., a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory -78WO 2017/023818

PCT/US2016/044922 region 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 regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS-responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the 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. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the propionate catabolism enzyme in the presence of ROS.

[0211] In some embodiments, 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. In some embodiments, 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. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0212] In some embodiments, 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). In some embodiments, 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 ROSresponsive regulatory region. In some embodiments, 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.

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PCT/US2016/044922 [0213] Thus, in some embodiments, 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.

[0214] In some embodiments, 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. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein. In some embodiments, the gene encoding the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the amino acid catabolism enzyme, propionate transporter, and/or propionate binding protein. In some embodiments, the gene encoding the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed on a chromosome.

[0215] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene encoding a particular propionate catabolism enzyme, propionate transporter, and/or propionate binding protein inserted at four different insertion sites. Alternatively, 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

-80WO 2017/023818

PCT/US2016/044922 propionate catabolism enzyme, propionate transporter, and/or propionate binding protein inserted at three different insertion sites.

[0216] In some embodiments, under conditions where the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about

2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300fold, 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.

[0217] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the propionate catabolism enzyme, propionate transporter, and/or propionate binding protein gene(s). 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. In some embodiments, 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 3545° 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).

[0218] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the propionate catabolism enzyme, propionate -81WO 2017/023818

PCT/US2016/044922 transporter, and/or propionate binding protein gene(s). 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. In some embodiments, 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, 6070° 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).

[0219] In other embodiments, the inducible promoter is a propionate responsive promoter. For example, the prpR promoter is a propionate responsive promoter. In one embodiment, the propionate responsive promoter comprises SEQ ID NO: 70.

Propionate Catabolism Enzymes and Propionate Catabolism Genes and Gene

Cassettes [0220] As used herein, 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.

[0221] As used herein, 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

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PCT/US2016/044922 and/or a metabolite thereof. For example, 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. For example, 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. Examples of 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), 2methylcitrate dehydratase (PrpD), propionyl-CoA carboxylase (pccB), Acetyl-/propionylcoenzyme A carboxylase (accAl), Methylmalonyl-CoA epimerase (mmcE), methylmalonylCoA mutase (mutA, and mutB), Acetoacetyl-CoA reductase (phaB), Polyhydroxyalkanoic acid (PHA) synthases, e.g.,encoded by phaC, and 3-ketothiolase (phaA), pet, and malonylcoenzyme A (malonyl-CoA) synthetase (ma.tB).

[0222] Functional deficiencies in these proteins result in the accumulation of propionate and/or methylmalonic acid or one or more of their metabolites in cells and tissues. 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 wildtype 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.

[0223] As used herein, 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

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CoA molecule. Enzymes involved in the catabolism of propionate are well known to those of skill in the art.

[0224] In humans, the major pathway for metabolizing propionyl-CoA involves the enzyme propionyl CoA carboxylase (PCC), which converts propionyl CoA to methylmalonyl CoA, and the methylmalonyl CoA mutase (MUT) enzyme then converts methylmalonyl CoA into succinylCoA (see, e.g., FIG. 5). 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 MM A, and severe nutritional deficiencies of Vitamin Bp can also result in MMA (Higginbottom et al., M. Engl. J. Med., 299(7):317-323, 1978). Other minor pathways are present in humans, but these pathways are insufficient to compensate for the absence of or mutations in the major pathway for propionyl CoA metabolism (see, e.g., FIG. 5). Thus, in some embodiments, 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).

[0225] For propionic acid to be consumed by any of the pathways or circuits of the present disclosure, it must first be activated to propionyl-CoA. This activation can be catalyzed by either propionyl-CoA synthetase (PrpE) or propionate CoA transferase (Pet).

Thus, in some embodiments, 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 (Pet). In some embodiments, 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). In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more copies of propionate CoA transferase (Pet) 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 (Pet), one or more copies of propionyl CoA carboxylase (PCC) and one or more copies of methylmalonyl CoA mutase (MUT).

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PCT/US2016/044922 [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. However, the inactivation pathway can be eliminated entirely through the deletion of the pka gene. In any of the embodiments described herein and elsewhere in the specification, the genetically engineered bacteria comprise a deletion of pka (Apka) to prevent the inactivation of PrpE. In some embodiments the deletion of pka results in greater activity of PrpE and downstream catabolic enzymes.

[0227] Pet converts propionate and acetyl-CoA to propionyl-CoA and acetate in a reversible reaction. In some embodiments, the genetically engineered bacteria comprise a gene encoding Pet for the generation of propionylCoA from propionate, e.g., as shown in FIG. 9B. In some embodiments, the genetically engineered bacteria comprise Pet in combination with or as a component of one or more of PHA and/or MMCA and/or 2MC pathway cassette(s).

[0228] In bacteria, PrpB, PrpC, and PrpD are capable of converting propionyl CoA into succinate and pyruvate, and PrpB, PrpC, PrpD, and PrpE are capable of converting propionate into succinate and pyruvate. Specifically, PrpE, a propionate-CoA ligase, converts propionate to propionyl CoA. PrpC, a 2-methylcitrate synthetase, then converts propionyl CoA to 2-methylcitrate. PrpD, a 2-methylcitrate dehydrogenase, then converts 2methylcitrate into 2-methyisocitrate, and PrpB, a 2-methylisocitrate lyase, converts 2methyisocitrate into succinate and pyruvate (see FIG. 19). Thus, in some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpB, PrpC, and PrpD. In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpB, PrpC, PrpD, and PrpE. In some embodiments, the engineered bacterium comprises two or more copies of a gene encoding any of the following: PrpB, PrpC, and PrpD, and combinations thereof. In some embodiments, the engineered bacterium comprises two or more copies of a gene encoding any of the following: PrpB, PrpC, PrpD, and PrpE, and combinations thereof.

[0229] In another bacterial pathway, the polyhydroxyalkanoate pathway, propionate is converted to propionyl-CoA by PrpE. Propionyl-CoA is then converted to 3-keto-valerylCoA by PhaA, which is then converted to 3-hydroxy-valeryl-CoA by PhaB. Finally, PhaC

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PCT/US2016/044922 converts 3-hydroxy-valeryl-CoA to PHV (see FIG. 10). Thus, in some embodiments, 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. For example, a circuit can be designed to express prpE, pccB, accAl, mmcE, mutA, and mutB (FIG. 15). In this circuit, PrpE converts propionate to propionyl-CoA, which is then converted to D-methylmalonyl-CoA by PccB and AccAl. D-methylmalonyl-CoA is then converted to F-methylmalonyl-CoA by MmcE, and MutA and MutB convert F-methylmalonyl CoA to succinyl-CoA. Alternatively, these genes can be split up into two circuits, i.e, prpE-accAl-pccB and mmcE-mutA-mutB, as indicated in FIG. 15. Thus, in some embodiments, the engineered bacterium comprises gene sequence(s) selected from: prpE, pccB, accAl, mmcE, mutA, and mutB. In some embodiments, the engineered bacterium comprises gene sequence(s) encoding one or more of the following: PrpE, PccB, AccAl, MmcE, MutA, and MutB. In another embodiment, 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.

[0230] 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. These enzymes catalyze the conversion of organic acids to acyl-CoA thioesters via a ping-pong mechanism, in which ATP and the organic acid are first converted to acyl-AMP with the release of pyrophosphate, followed by coenzyme A binding, displacement of AMP, and release of the acyl-CoA product (see, e.g., Crosby et al., Structure-Guided Expansion of the Substrate Range of Methylmalonyl Coenzyme A Synthetase (MatB) of Rhodopseudomonas palustris; Appl. Environ. Microbiol. September 2012 vol. 78 no. 18 6619-6629, and references therein). MatB converts malonate to malonylCoA in two steps according to this mechanism via a malonyl-AMP intermediate, and similarly also converts methylmalonate to methylmalonyl-CoA.

[0231] 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 -86WO 2017/023818

PCT/US2016/044922 the MMCA pathway (e.g., through mmcE, mutA, and mutB). Alternatively, methylmalonylCoA can be converted to propionylCoA. This reaction may be catalyzed by the AccAl/PccB complex, which is encoded by a genetic circuit of the disclosure. The AccAl/pccB complex catalyzes the reversible conversion of propionylCoA to methylmalonylCoA, as described herein. Once methylmalonylCoA is converted to propionylCoA, any of the propionate catabolism enzymes encoded by the genetic circuits described herein, e.g., PHA, MMCA, and/or 2MC circuits, are suitable for further catalysis, resulting in an inert product. Thus, in any of the embodiments described herein and elsewhere in the specification, the engineered bacterium may further comprise gene sequence(s) encoding MatB.

[0232] In some embodiments of the disclosure, one or more gene(s) or gene cassette(s) comprise MatB, e.g., MatB derived from Rhodopseudomonas palustris. In some embodiments of the disclosure, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) comprising MatB, e.g., MatB derived from Rhodopseudomonas palustris. In a non-limiting example, 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.

[0233] 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 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 embodiments, MatB part of an operon comprising one or more gene(s) or gene cassette(s) encoding one or more propionate catabolism enzymes described herein.

[0234] In some embodiments, 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.

[0235] In some embodiments, the genetically engineered bacteria encode one or more of MatB, AcclA, and PccB. In some embodiments, the genetically engineered bacteria encode MatB, AcclA, and PccB. In some embodiments, a genetic circuit encoded by the genetically engineered bacteria comprises MatB, AcclA, and PccB. In some embodiments,

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PCT/US2016/044922 the genetically engineered bacteria encode MatB, AcclA, and PccB, and mmcE, mutA and mutB. In some embodiments, the genetically engineered bacteria encode MatB, AcclA, and PccB, and mmcE, mutA and mutB and further prpE. In some embodiments, the genetically engineered bacteria encode MatB, AcclA, 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. Nonlimiting 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. In other non-limiting examples, the one or more gene cassettes may be organized as follows; MatB-mmcE-mutA-mutB; MatB-AcclA-PccB and mmcE-mutA-mutB, alone or in combination with PPHA and/or 2MC pathway cassettes; PrpE-MatB-AcclA-PccB and mmcE-mutA-mutB, alone or in combination with PPHA and/or 2MC pathway cassettes.

[0236] In one embodiment, 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 embodiment, 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.

[0237] In another embodiment, 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. In another embodiment, 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. In one embodiment, 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. In one embodiment, 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.

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PCT/US2016/044922 [0238] Enzymes involved in the catabolism of propionate may be expressed or modified in the bacteria in order to enhance catabolism of propionate. Specifically, when the heterologous propionate catabolism gene or gene cassette is 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. Thus, 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).

[0239] In some embodiments, the expression of the propionate catabolism gene cassette decreases the levels of one or more propionic acidemia and/or methylmalonic acidemia biomarkers. In some embodiments, 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. In one embodiment, 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. In one embodiment, 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 methylmalonate. In one embodiment, 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.

[0240] In one embodiment, 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.

[0241] In one embodiment, the engineered bacterial cell comprises at least one heterologous gene or gene cassette encoding at least one propionate catabolism enzyme. In some embodiments, the disclosure provides a bacterial cell that comprises at least one -89WO 2017/023818

PCT/US2016/044922 heterologous gene or gene cassette encoding at least one propionate catabolism enzyme operably linked to a first promoter. In one embodiment, 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. In another embodiment, the bacterial cell comprises more than one copy of a native gene or gene cassette encoding one or more propionate catabolism enzyme(s). In yet another embodiment, 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. In one embodiment, 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). In one embodiment, the bacterial cell comprises multiple copies of a gene or gene cassette encoding one or more propionate catabolism enzyme(s). In one embodiment, a gene cassette may comprise one or more native and one or more non-native or heterologous genes.

[0242] Multiple distinct propionate catabolism enzymes are known in the art. In some embodiments, the propionate catabolism enzyme is encoded by at least one gene encoding at least one propionate catabolism enzyme derived from a bacterial species. In some embodiments, 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. In some embodiments, 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 embodiment, a propionate catabolism enzyme is encoded by a gene derived from a human.

In one embodiment, 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 -90WO 2017/023818

PCT/US2016/044922 thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longurn, 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, Elelicobacter mustelae, Klebsiella pneumonia, Klebsiella variicola, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Eactococcus 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.

[0243] In some embodiments, the gene encoding prpE is derived from E. coli. In some embodiments, the gene encoding accAl is derived from Streptopmyces coelicolor. In some embodiments, the gene encoding pccB is derived from E. coli. In some embodiments, the gene encoding mmcE is derived from Propionibcteriumfreudenreichii. In some embodiments, the gene encoding mutA is derived from Propionibcterium freudenreichii. In some embodiments, the gene encoding mutB is derived from Propionibcterium freudenreichii. In some embodiments, 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

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PCT/US2016/044922 phaC is derived from Acinetobacter sp RA3849. In some embodiments, the gene encoding phaA is derived from Acinetobacter sp RA3849.

[0244] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme has been codon-optimized for use in the engineered bacterial cell. In one embodiment, 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. When 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). Thus, 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 B12 deficiency.

[0245] 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). As used herein, 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. For example, 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.

For example, 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. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate catabolism enzyme functional variant. In another embodiment, the engineered bacterial cell comprises a heterologous gene or gene cassette encoding a propionate catabolism enzyme functional fragment.

[0246] 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. For example, propionate -92WO 2017/023818

PCT/US2016/044922 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. In another example, 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. For example, 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). Alternatively, propionate levels can be determined using high-pressure liquid chromatography (HPLC). For example, 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., 2003,7. Bacteriol., 185(9):2802-2810).

[0247] In mammals, 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.

[0248] As used herein, the term “percent (%) sequence identity” or “percent (%) identity,” also including homology, is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

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PCT/US2016/044922 [0249] 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. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Fys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Feu, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).

[0250] In some embodiments, 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. In one embodiment, 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.

[0251] In one embodiment, 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. In another embodiment, 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%,

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PCT/US2016/044922

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. In another embodiment, 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.

[0252] In one embodiment, 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. In another embodiment, 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. In another embodiment, 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.

[0253] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is prpD. prpD encodes PrpD, a 2-methylcitrate dehydrogenase.

Accordingly, in one embodiment, 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. Accordingly, in one embodiment, the prpD gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, -95WO 2017/023818

PCT/US2016/044922 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.

[0254] In one embodiment, 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. In another embodiment, 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. In another embodiment, 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.

[0255] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is phaB. phaB encodes PhaB, a acetoacetyl-CoA reductase.

Accordingly, in one embodiment, 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.

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PCT/US2016/044922 [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. Accordingly, in one embodiment, 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.

[0257] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, the phaA gene comprises a sequence which encodes SEQ ID NO: 28. In yet another embodiment the phaA gene consists of a sequence which encodes SEQ ID NO: 28.

[0258] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is pccB. pccB encodes PccB, a propionyl CoA carboxylase.

Accordingly, in one embodiment, 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.

[0259] In one embodiment, 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

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PCT/US2016/044922 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.

[0260] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is accAl. accAl encodes AccAl, an acetyl CoA carboxylase. Accordingly, in one embodiment, the accAl gene has at least about 80% identity with SEQ ID NO: 38. In one embodiment, the accAl gene has at least about 90% identity with SEQ ID NO: 38. In one embodiment, the accAl gene has at least about 95% identity with SEQ ID NO: 38. In one embodiment, the accAl 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 accAl gene comprises the sequence of SEQ ID NO: 38. In yet another embodiment, the accAl gene consists of the sequence of SEQ ID NO: 38.

[0261] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is accAl. accAl encodes AccAl, an acetyl CoA carboxylase. Accordingly, in one embodiment, the accAl gene has at least about 80% identity with SEQ

ID NO: 104. In one embodiment, the accAl gene has at least about 90% identity with SEQ

ID NO: 104. In one embodiment, the accAl gene has at least about 95% identity with SEQ

ID NO: 104. In one embodiment, the accAl 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 accAl gene comprises the sequence of SEQ ID NO: 104.

In yet another embodiment, the accAl gene consists of the sequence of SEQ ID NO: 104.

[0262] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is mmcE. mmcE encodes MmcE, a methylmalonyl-CoA mutase. Accordingly, in one embodiment, 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.

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PCT/US2016/044922 [0263] In one embodiment, 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.

[0264] In one embodiment, 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. 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: 33. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO: 33. In yet another embodiment, the mutA gene consists of the sequence of SEQ ID NO: 33.

[0265] In one embodiment, 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.

[0266] In one embodiment, 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. In one embodiment, the mutB gene has at least about 85%, 86%, 87%,

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88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 34. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO: 34. In yet another embodiment, the mutB gene consists of the sequence of SEQ ID NO: 34.

[0267] In one embodiment, 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. In one embodiment, 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. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO: 112. In yet another embodiment, the mutB gene consists of the sequence of SEQ ID NO: 112.

[0268] In one embodiment, 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.

Accordingly, in one embodiment, 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. In another embodiment, the prpE comprises a sequence which encodes SEQ ID NO: 71. In yet another embodiment, prpE consists of a sequence which encodes SEQ ID NO: 71.

[0269] In one embodiment, the at least one propionate catabolism enzyme is phaA. Accordingly, in one embodiment, phaB has at least about 80% identity with SEQ ID NO:

137. In one embodiment, phaA has at least about 90% identity with SEQ ID NO: 175. In another embodiment, phaA has at least about 95% identity with SEQ ID NO: 137. Accordingly, in one embodiment, 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. In another embodiment, phaA comprises a sequence which encodes SEQ ID NO: 137. In yet another embodiment phaA consists of a sequence which encodes SEQ ID NO: 137.

[0270] In one embodiment, 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

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PCT/US2016/044922 another embodiment, phaB has at least about 95% identity with SEQ ID NO: 135. Accordingly, in one embodiment, 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. In another embodiment, phaB comprises a sequence which encodes SEQ ID NO: 135. In yet another embodiment phaB consists of a sequence which encodes SEQ ID NO: 135.

[0271] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, phaC comprises a sequence which encodes SEQ ID NO: 136. In yet another embodiment phaC consists of a sequence which encodes SEQ ID NO: 136.

[0272] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, mmcE comprises a sequence which encodes SEQ ID NO: 132. In yet another embodiment mmcE consists of a sequence which encodes SEQ ID NO: 132.

[0273] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, mutA comprises a sequence which encodes SEQ ID NO: 133. In yet another embodiment mutA consists of a sequence which encodes SEQ ID NO: 133.

[0274] In one embodiment, 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.

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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.

[0275] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, accA comprises a sequence which encodes SEQ ID NO: 130. In yet another embodiment the accA consists of a sequence which encodes SEQ ID NO: 130.

[0276] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, pccB comprises a sequence which encodes SEQ ID NO: 131. In yet another embodiment, pccB consists of a sequence which encodes SEQ ID NO: 131.

[0277] In one embodiment, the at least one propionate catabolism enzyme is prpC. Accordingly, in one embodiment, prpC has at least about 80% identity with SEQ ID NO: 74. In one embodiment, prpC has at least about 90% identity with SEQ ID NO: 74. In another embodiment, prpC has at least about 95% identity with SEQ ID NO: 74. Accordingly, in one embodiment, 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. In another embodiment, prpC comprises a sequence which encodes SEQ ID NO: 74. In yet another embodiment, prpC consists of a sequence which encodes SEQ ID NO: 74.

[0278] In one embodiment, the at least one propionate catabolism enzyme is prpD.

Accordingly, in one embodiment, prpD has at least about 80% identity with SEQ ID NO: 77.

In one embodiment, prpD has at least about 90% identity with SEQ ID NO: 77. In another embodiment, prpD has at least about 95% identity with SEQ ID NO: 77. Accordingly, in one embodiment, prpD has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,

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94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 77. In another embodiment, prpD comprises a sequence which encodes SEQ ID NO: 77. In yet another embodiment, prpD consists of a sequence which encodes SEQ ID NO: 77.

[0279] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is MatB. MatB encodes Malonyl-coenzyme A (malonyl-CoA) synthetase (MatB). Accordingly, in one embodiment, the MatB gene has at least about 80% identity with SEQ ID NO: 141. Accordingly, in one embodiment, the MatB gene has at least about 90% identity with SEQ ID NO: 141. Accordingly, in one embodiment, the MatB gene has at least about 95% identity with SEQ ID NO: 141. Accordingly, in one embodiment, 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. In another embodiment, the MatB gene comprises the sequence of SEQ ID NO: 141. In yet another embodiment the MatB gene consists of the sequence of SEQ ID NO: 141.

[0280] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, matB comprises a sequence which encodes SEQ ID NO: 140. In yet another embodiment, matB consists of a sequence which encodes SEQ ID NO: 140.

[0281] In one embodiment, the at least one propionate catabolism enzyme is prpB. Accordingly, in one embodiment, prpB has at least about 80% identity with SEQ ID NO: 80. In one embodiment, prpB has at least about 90% identity with SEQ ID NO: 80. In another embodiment, prpB has at least about 95% identity with SEQ ID NO: 80. Accordingly, in one embodiment, 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. In another embodiment, prpB comprises a sequence which encodes SEQ ID NO: 80. In yet another embodiment, prpB consists of a sequence which encodes SEQ ID NO: 80.

[0282] In one embodiment, any combination of propionate catabolism enzymes that effectively reduce the level of propionate and/or a metabolite thereof can be used. In one embodiment, any combination of propionate catabolism enzymes that effectively reduce levels of propionate, propionyl CoA, and/or methylmalonyl CoA in a subject can be used. In

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PCT/US2016/044922 one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is prpBCD. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is prpBCDE. Using all four heterologous genes, for example, prpBCDE, is not necessary but allows excess propionate to be converted into succinate and pyruvate, feeding the Krebs cycle and benefiting the bacteria by increasing their growth. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is prpE, pccB, accAl, mmcE, mutA, and mutB. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is prpE, pccB, and accAl under the control of a first inducible promoter, and mmcE, mutA, and mutB under the control of a second inducible promoter. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is prpE, phaB, phaC, and phaA.

[0283] In one embodiment, 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:

OR SEQ ID NO: 84. Accordingly, in one embodiment, the prpBCD operon has at least about 95% identity with SEQ ID NO: 138. In another embodiment, 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. In another embodiment, 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.

[0284] In one embodiment, 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 -104WO 2017/023818

PCT/US2016/044922 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. In another embodiment, 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. In another embodiment, 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.

[0285] In one embodiment, the propionate catabolism gene cassette comprises phaBCA. Accordingly, in one embodiment, the phaBCA operon has at least about 80% identity with SEQ ID NO: 139. In one embodiment, the phaBCA operon has at least about 90% identity with SEQ ID NO: 139. In one embodiment, the phaBCA operon has at least about 95% identity with SEQ ID NO: 139. In one embodiment, 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. In another embodiment, 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.

[0286] In one embodiment, the propionate catabolism gene cassette comprises phaBCA. Accordingly, in one embodiment, the phaBCA operon has at least about 80% identity with SEQ ID NO: 102. In one embodiment, the phaBCA operon has at least about 90% identity with SEQ ID NO: 102. In one embodiment, the phaBCA operon has at least about 95% identity with SEQ ID NO: 102. In one embodiment, 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. In another embodiment, the phaBCA operon comprises the sequence of SEQ ID NO: 102. In another embodiment, the phaBCA operon

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PCT/US2016/044922 consists of the sequence of SEQ ID NO: 102. In one embodiment, the propionate catabolism gene cassette comprises prpE and phaBCA.

[0287] In one embodiment, the propionate catabolism gene cassette comprises prpEphaBCA. Accordingly, in one embodiment, the prpE-phaBCA operon has at least about 80% identity with SEQ ID NO: 24. In one embodiment, the prpE-phaBCA operon has at least about 90% identity with SEQ ID NO: 24. In one embodiment, the prpE-phaBCA operon has at least about 95% identity with SEQ ID NO: 24. In one embodiment, 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. In another embodiment, the prpEphaBCA operon comprises the sequence of SEQ ID NO: 24. In another embodiment, the prpE-phaBCA operon consists of the sequence of SEQ ID NO: 24.

[0288] In one embodiment, the propionate catabolism gene cassette comprises prpE, pccB, accAl, mmcE, mutA, and mutB. Accordingly, in one embodiment, the prpE-pccBaccAl-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-accAl-mmcE-mutA-mutB operon has at least about 90% identity with a combination of SEQ ID NO: 37 and 31. In one embodiment, the prpE-pccB-accAl-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-accAl 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-accAl -mmcE-mutA-mutB operon comprises the sequence of a combination of SEQ ID NO: 37 and 31. In another embodiment, the prpEpccB-accAl-mmcE-mutA-mutB operon consists of the sequence of a combination of SEQ ID NO: 37 and 31.

[0289] In one embodiment, the propionate catabolism gene cassette comprises prpE, pccB, and accAl. Accordingly, in one embodiment, the prpE-pccB-accAl operon has at least about 80% identity with SEQ ID NO: 37. In one embodiment, the prpE-pccB-accAl operon has at least about 90% identity with SEQ ID NO: 37. In one embodiment, the prpE-pccBaccAl operon has at least about 95% identity with SEQ ID NO: 37. In one embodiment, the prpE-pccB-accAl 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. In another embodiment, the prpE-pccB-accAl operon comprises the sequence of SEQ ID NO: 37. In

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PCT/US2016/044922 another embodiment, the prpE-pccB-accAl operon consists of the sequence of SEQ ID NO:

37.

[0290] In one embodiment, 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 mmcEmutA-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. In one embodiment, 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. In another embodiment, the mmcEmutA-mutB operon comprises the sequence of a combination of SEQ ID NO: 31. In another embodiment, the mmcE-mutA-mutB operon consists of the sequence of a combination of SEQ ID NO: 31.

[0291] In one embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is directly operably linked to a first promoter. In another embodiment, the at least one gene encoding the at least one propionate catabolism enzyme is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the at least one gene encoding the propionate catabolism enzyme in nature.

[0292] In some embodiments, 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. In one embodiment, 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. Inducible promoters are described in more detail infra.

[0293] 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 -107WO 2017/023818

PCT/US2016/044922 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. In yet another embodiment, 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. In yet another embodiment, 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. In yet another embodiment, 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.

[0294] In some embodiments, 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.

[0295] In some embodiments, 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. Thus, in some embodiments, it may be advantageous to increase propionate transport into the cell, thereby enhancing propionate catabolism. Furthermore, in some embodiments that incorporate 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 -108WO 2017/023818

PCT/US2016/044922 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. In alternate embodiments, the importer of propionate is used in conjunction with a high-copy plasmid.

[0296] Deacylation of propioonylated PrpE (PrpE^Plj by CobB, a NAD-dependent deacylase, allows bacterial cells to catabolize propionate. Thus, in one embodiment, 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). In one embodiment, 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. Accordingly, in one embodiment, 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.

[0297] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, CobB comprises a sequence which encodes SEQ ID NO: 113. In yet another embodiment, CobB consists of a sequence which encodes SEQ ID NO: 113.

[0298] In another embodiment, the engineered bacterial cell comprising a heterologous cobB gene further comprises a genetic modification in the pka gene. Pka, a protein lysine acetyltransferase, renders PrpE in the propionylated form (PrpE^Plj unable to metabolize propionate. Therefore, genetic modification of the pka gene (SEQ ID NO: 116) which renders it functionally inactive enhances the ability of the bacterial cells to catabolize propionate.

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Transporter (Importer) of Propionate [0299] The uptake of propionate into bacterial cells typically occurs via passive diffusion (see, for example, Kell etal., 1981, Biochem. Biophys. Res. Commun., 9981-9988). However, the active import of propionate is also mediated by proteins well known to those of skill in the art. For example, a bacterial transport system for the update of propionate in Corynebacterium glutamicum named MctC (monocarboxylic acid transporter) is known (see, for example, Jolkver et al., 2009, J. Bacteriol., 191(3):940-948). The putP_6 propionate transporter from Virgibacillus species (UniProt A0A024QGU1) has also been identified.

[0300] Propionate transporters, e.g., propionate importers, may be expressed or modified in the bacteria in order to enhance propionate transport into the cell. Specifically, when 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. Thus, 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 Bn deficiencies. In one embodiment, the bacterial cell comprises a heterologous gene encoding transporter of propionate. In one embodiment, 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.

[0301] Thus, in some embodiments, 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. In one embodiment, the first promoter and the second promoter are separate copies of the same promoter. In another embodiment, the first promoter and the second promoter are different promoters.

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PCT/US2016/044922 [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. In yet another embodiment, 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. In one embodiment, 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. In one embodiment, the bacterial cell comprises multiple copies of the at least one heterologous gene encoding a transporter of propionate.

[0303] In some embodiments, 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, Clostridium perfringens, Escherichia coli, Lactobacillus delbrueckii, Pseudomonas aeruginosa, Salmonella typhimurium, Virgibacillus, or Staphylococcus aureus. In some embodiments, the bacteria is a Virgibacillus. In some embodiments, the bacterial is a Corynebacterium. In one embodiment, the bacteria is C. glutamicum. In another embodiment, the bacteria is C. diphtheria. In another embodiment, the bacteria is C. efficiens. In another embodiment, 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.

[0304] The present disclosure further comprises genes encoding functional fragments of a transporter of propionate or functional variants of a transporter of propionate. As used herein, 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. For example, 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

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PCT/US2016/044922 derived. In one embodiment, 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.

[0305] 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. For example, 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. For example, 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). Alternatively, samples can be analyzed for propionate import using high-pressure liquid chromatography (HPLC). For example, 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).

[0306] In one embodiment the 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.

[0307] 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.

[0308] In some embodiments, 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. In some embodiments, the at least one gene encoding a transporter of propionate is mutagenized; mutants exhibiting decreased propionate transport

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PCT/US2016/044922 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.

[0309] In one embodiment, the propionate importer is MctC. In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, the mctC gene comprises the sequence of SEQ ID NO: 88. In yet another embodiment the mctC gene consists of the sequence of SEQ ID NO: 88.

[0310] In one embodiment, 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.

[0311] In another embodiment, the propionate importer is PutP_6. In one embodiment, the putP_6 gene has at least about 80% identity to SEQ ID NO: 90. Accordingly, in one embodiment, 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.

[0312] In one embodiment, 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.

Accordingly, in one embodiment, PutP_6 has at least about 85%, 86%, 89%, 88%, 89%,

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90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 89. In another embodiment, PutP_6 comprises a sequence which encodes SEQ ID NO: 89. In yet another embodiment, PutP_6 consists of a sequence which encodes SEQ ID NO: 89.

[0313] Other 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. In one embodiment, the propionate importer comprises the mctBC genes from C. glutamicum. In another embodiment, 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 cel091 and cel092 genes from C. efficiens. In another embodiment, the propionate importer comprises the scol822 and scol823 genes from S. coelicolor. In another embodiment, the propionate importer comprises the scol218 and scol219 genes from S. coelicolor. In another embodiment, the propionate importer comprises the cel091 and sco5827 genes from S. coelicolor. In another embodiment, the propionate importer comprises the m_5160, m_5161, m_5165, and m_5166 genes from M. smegmatis. In another embodiment, the propionate importer comprises the nfa 17930, nfa 17940, nfa 17950, and nfa 17960 genes from N. farcinica. In another embodiment, the propionate importer comprises the actP and yjcH genes from E. coli. In another embodiment, the propionate importer comprises the ywcB and ywcA genes from B. subtilis.

[0314] In some embodiments, 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. In some embodiments, 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.

[0315] In some embodiments, 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 -114WO 2017/023818

PCT/US2016/044922 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.

[0316] In one embodiment, the promoter is not operably linked with the at least one gene encoding a transporter of propionate in nature. In some embodiments, the at least one gene encoding the importer of propionate is controlled by its native promoter. In some embodiments, the at least one gene encoding the importer of propionate is controlled by an inducible promoter. In some embodiments, the at least one gene encoding the importer of propionate is controlled by a promoter that is stronger than its native promoter. In some embodiments, the at least one gene encoding the importer of propionate is controlled by a constitutive promoter.

[0317] In another embodiment, the promoter is an inducible promoter. Inducible promoters are described in more detail infra.

[0318] In one embodiment, 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. In yet another embodiment, 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.

[0319] In some embodiments, 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. In one embodiment, 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 -115WO 2017/023818

PCT/US2016/044922 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. In alternate embodiments, 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. In one embodiment, 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.

[0320] In one embodiment, when the importer of propionate is expressed in the engineered bacterial cells, 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. In yet another embodiment, when the importer of propionate is expressed in the engineered bacterial cells, 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.

Exporters of Succinate [0321] 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. For example, a succinate exporter in Corynebacterium glutamicum is known as SucEl. SucEl is a membrane protein belonging to the aspartate:alanine exchanger (AAE) family (see, for example, Fukui etal., 2011, J. Bacteriol., 154(1):25-34). The DcuC succinate exporter from

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E. coli has also been identified (see, for example, Cheng et al., 2013, J. Biomed. Res. Int.,

2013:ID 538790).

[0322] Succinate transporters, e.g., succinate exporters, may be expressed or modified in the bacteria in order to enhance succinate export out of the cell. Specifically, when the exporter of succinate is expressed in the engineered bacterial cells, 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. In one embodiment, the bacterial cell comprises a heterologous gene encoding an exporter of succinate. In one embodiment, 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.

[0323] Thus, in some embodiments, 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. In some embodiments, the at least one heterologous gene encoding an exporter of succinate is operably linked to the first promoter. In another embodiment, the at least one heterologous gene encoding the at least one propionate catabolism enzyme operably is linked to a first promoter, and the heterologous gene encoding an exporter of succinate is operably linked to a second promoter. In one embodiment, the first promoter and the second promoter are separate copies of the same promoter. In another embodiment, the first promoter and the second promoter are different promoters.

[0324] In one embodiment, the bacterial cell comprises at least one gene encoding an exporter of succinate from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one native gene encoding an exporter of succinate. In some embodiments, the at least one native gene encoding an exporter of succinate is not modified. In another embodiment, the bacterial cell comprises more than one copy of at least one native gene encoding an exporter of succinate. In yet another embodiment, 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. In one embodiment, 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.

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PCT/US2016/044922 [0325] In some embodiments, 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 typhimurium, Klebsiella pneumoniae, Serratia plymuthica, Enterobacter cloacae, Bacillus subtilis, Bacillus anthracis, bacillus lichenformis, and Saccharomyces cerevisiae. In some embodiments, the exporter of succinate is derived from Corynebacterium. In one embodiment, 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.

[0326] The present disclosure further comprises genes encoding functional fragments of an exporter of succinate or functional variants of an exporter of succinate. As used herein, 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. For example, 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. In one embodiment, the engineered bacterial cell comprises at least one heterologous gene encoding a functional fragment of an exporter of succinate. In another embodiment, the engineered bacterial cell comprises at least one heterologous gene encoding a functional variant of an exporter of succinate.

[0327] In some embodiments, the genetically engineered bacteria further comprise a mutation or deletion in one or more succinate importers, e.g., Dct, DctC, ybhl or ydjN. In some embodiments, succinate dehydrogenase (SUCDH) may be mutated or deleted. Without wishing to be bound by theory, such mutations may decrease intracellular succinate concentrations and increase the flux through propionate catabolism pathways.

[0328] Assays for testing the activity of an exporter of succinate, an exporter of succinate functional variant, or an exporter of succinate functional fragment are well known to one of ordinary skill in the art. For example, 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

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PCT/US2016/044922 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.

[0329] In one embodiment 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.

[0330] 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.

[0331] In some embodiments, 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. In some embodiments, 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.

[0332] In one embodiment, the succinate exporter is DcuC. In one embodiment, 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. 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: 49 In another embodiment, the dcuC gene comprises the sequence of SEQ ID NO: 49. In yet another embodiment the dcuC gene consists of the sequence of SEQ ID NO:70.

[0333] In one embodiment, 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. Accordingly, in one embodiment, 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. In

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PCT/US2016/044922 another embodiment, DcuC comprises a sequence which encodes SEQ ID NO: 129. In yet another embodiment, DcuC consists of a sequence which encodes SEQ ID NO: 129.

[0334] In one embodiment, the succinate exporter is DcuC. In one embodiment, 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.

Accordingly, in one embodiment, 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.

[0335] In one embodiment, 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. Accordingly, in one embodiment, 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. In another embodiment, DcuC comprises a sequence which encodes SEQ ID NO: 117. In yet another embodiment, DcuC consists of a sequence which encodes SEQ ID NO: 117.

[0336] In another embodiment, the succinate exporter is SucEl. In one embodiment, the sucEl gene has at least about 80% identity to SEQ ID NO: 46. Accordingly, in one embodiment, the sucEl gene has at least about 90% identity to SEQ ID NO: 46.

Accordingly, in one embodiment, the sucEl gene has at least about 95% identity to SEQ ID NO: 46. Accordingly, in one embodiment, the sucEl 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 sucEl gene comprises the sequence of SEQ ID NO: 46. In yet another embodiment the sucEl gene consists of the sequence of SEQ ID NO: 46.

[0337] In another embodiment, the succinate exporter is SucEl. In one embodiment, the sucEl gene has at least about 80% identity to SEQ ID NO: 120. Accordingly, in one embodiment, the sucEl gene has at least about 90% identity to SEQ ID NO: 120.

Accordingly, in one embodiment, the sucEl gene has at least about 95% identity to SEQ ID

NO: 120. Accordingly, in one embodiment, the sucEl 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 sucEl gene comprises the sequence of SEQ ID NO: -120WO 2017/023818

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120. In yet another embodiment the sucEl gene consists of the sequence of SEQ ID NO:

120.

[0338] In one embodiment, the at least one succinate exporter is sucEl. Accordingly, in one embodiment, sucEl has at least about 89% identity with SEQ ID NO: 128. In one embodiment, sucEl has at least about 90% identity with SEQ ID NO: 128. In another embodiment, sucEl has at least about 95% identity with SEQ ID NO: 128. Accordingly, in one embodiment, sucEl 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, sucEl comprises a sequence which encodes SEQ ID NO: 128. In yet another embodiment, sucEl consists of a sequence which encodes SEQ ID NO: 128. In another embodiment, the sucEl has at least about 89% identity with SEQ ID NO: 119. In one embodiment, sucEl has at least about 90% identity with SEQ ID NO: 119. In another embodiment, sucEl has at least about 95% identity with SEQ ID NO: 119. Accordingly, in one embodiment, sucEl 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, sucEl comprises a sequence which encodes SEQ ID NO: 119. In yet another embodiment, sucEl consists of a sequence which encodes SEQ ID NO: 119.

[0339] In some embodiments, 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. In some embodiments, the at least one heterologous gene encoding an exporter of succinate is operably linked to the first promoter. In other embodiments, the at least one heterologous gene encoding an exporter of succinate is operably linked to a second promoter. In one embodiment, the at least one gene encoding an exporter of succinate is directly operably linked to the second promoter. In another embodiment, the at least one gene encoding an exporter of succinate is indirectly operably linked to the second promoter.

[0340] In some embodiments, 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. In some embodiments, 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. In some embodiments, at least one gene encoding an exporter of succinate and the propionate catabolism enzyme are divergently transcribed from a promoter -121WO 2017/023818

PCT/US2016/044922 region. In some embodiments, 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.

[0341] In one embodiment, the promoter is not operably linked with the at least one gene encoding an exporter of succinate in nature. In some embodiments, the at least one gene encoding the exporter of succinate is controlled by its native promoter. In some embodiments, 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.

[0342] In another embodiment, the promoter is an inducible promoter. Inducible promoters are described in more detail infra.

[0343] In one embodiment, the at least one gene encoding an exporter of succinate is located on a plasmid in the bacterial cell. In another embodiment, the at least one gene encoding an exporter of succinate is located in the chromosome of the bacterial cell. In yet another embodiment, 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. In yet another embodiment, 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. In yet another embodiment, 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.

[0344] In some embodiments, 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. In one embodiment, 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 -122WO 2017/023818

PCT/US2016/044922 promoter. In alternate embodiments, 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. In one embodiment, 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.

[0345] In one embodiment, 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. In yet another embodiment, 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 [0346] As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Fin, 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).

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PCT/US2016/044922 [0347] 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.

[0348] An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is an oligonucleotide synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, ser A or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF,flhD, metB, metC, proAB, and thil, as long as the corresponding wild-type gene product is not produced in the bacteria.

[0349] Table 7 lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.

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

Auxotroph

Amino Acid	Oligonucleotide	Cell Wall
cysE	thyA	dapA
glnA	uraA	dapB
ilvD		dapD
leuB		dapE
lysA		dapF
serA

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PCT/US2016/044922

metA
glyA
hisB
ilvA
pheA
proA
thrC
trpC
tyrA

[0350] Table 8 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.

Table 8. Survival of amino acid auxotrophs in the mouse gut

Gene	AA Auxotroph	Pre-Gavage	24 hours	48 hours
argA	Arginine	Present	Present	Absent
cysE	Cysteine	Present	Present	Absent
glnA	Glutamine	Present	Present	Absent
giyA	Glycine	Present	Present	Absent
hisB	Histidine	Present	Present	Present
ilvA	Isoleucine	Present	Present	Absent
leuB	Leucine	Present	Present	Absent
lysA	Lysine	Present	Present	Absent
metA	Methionine	Present	Present	Present
pheA	Phenylalanine	Present	Present	Present
proA	Proline	Present	Present	Absent
serA	Serine	Present	Present	Present
thrC	Threonine	Present	Present	Present
trpC	Tryptophan	Present	Present	Present
tyrA	Tyrosine	Present	Present	Present
ilvD	V aline/Isoleucin e/Leucine	Present	Present	Absent
thyA	Thiamine	Present	Absent	Absent
uraA	Uracil	Present	Absent	Absent
flhD	FlhD	Present	Present	Present

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PCT/US2016/044922 [0351] For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0352] Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0353] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0354] In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph.

Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease

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PCT/US2016/044922 the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

[0355] Other examples of 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, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, om, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, mpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, ripB, leuS, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabl, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

[0356] In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, ’’ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

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PCT/US2016/044922 [0357] In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G.

[0358] In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

[0359] In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some -128WO 2017/023818

PCT/US2016/044922 embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), andpheS (F125G, P183T, P184A, R186A, and I188L).

[0360] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.

[0361] In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (see Wright et al., supra).

Genetic Regulatory Circuits [0362] In some embodiments, the genetically engineered bacteria comprise multilayered 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. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.

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PCT/US2016/044922 [0363] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the payload is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the payload is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

[0364] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNRresponsive 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.

[0365] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a first

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PCT/US2016/044922 regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNRresponsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.

[0366] Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, Lacl, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

[0367] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNRresponsive promoter, and a second gene or gene cassette for producing a payload. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the payload. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing payload translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.

[0368] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein;

a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a

FNR-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive

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PCT/US2016/044922 promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed.

[0369] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a payload operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3’ to 5’) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5’ to 3’). In the presence of oxygen, FNR does not bind the FNRresponsive promoter, the recombinase is not expressed, the payload remains in the 3’ to 5’ orientation, and no functional payload is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the payload is reverted to the 5’ to 3’ orientation, and functional payload is produced.

[0370] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload.

The third gene encoding the T7 polymerase is inverted in orientation (3’ to 5’) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5’ to 3’). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3’ to 5’ orientation, and

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PCT/US2016/044922 the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNRresponsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5’ to 3’ orientation, and the payload is expressed.

Kill Switches [0371] In some embodiments, the genetically engineered bacteria also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329, each of which are expressly incorporated herein by reference in their entireties). The kill switch is intended to actively kill engineered microbes in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

[0372] Bacteria engineered with kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease or disorder may also be programmed to die at a specific time after the expression and delivery of a heterologous gene, genes or gene cassette(s), for example, a therapeutic gene(s) or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following expression of the propionate catabolism enzyme cassette(s) and/or gene(s) present in the engineered bacteria.

In some embodiments, the kill switch is activated in a delayed fashion following expression of the heterologous gene(s) or gene cassette(s), for example, after the production of the corresponding protein(s) or molecule(s). Alternatively, the bacteria may be engineered to die after the bacteria has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject).

[0373] 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),

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PCT/US2016/044922 translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl β-D-l-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010). In some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of a heterologous gene(s) or gene cassette(s). In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of a heterologous gene(s) or gene cassette(s).

[0374] 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).

[0375] Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the engineered bacterial cell is no longer viable.

[0376] In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental -134WO 2017/023818

PCT/US2016/044922 condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[0377] In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

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PCT/US2016/044922 [0378] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase. Accordingly, in one embodiment, the disclosure provides at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 recombinases that can be used serially.

[0379] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed engineered bacterial cell is not viable after the first essential gene is excised.

[0380] In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the wherein the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.

[0381] In one embodiment, the first excision enzyme is Xisl. In one embodiment, the first excision enzyme is Xis2. In one embodiment, the first excision enzyme is Xisl, and the second excision enzyme is Xis2.

[0382] In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

[0383] In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxbl, PhiC31, TP901, Bxbl, PhiC31, TP901, HK022, HP1, R4,

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Inti, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, IntlO, Intll, Intl2, Intl3, Intl4, Intl5, Intl6, Intl7, Intl8, Intl9, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.

[0384] In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill-switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) are described herein. The disclosure provides engineered bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the engineered bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxin gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arabinose system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

[0385] Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an antitoxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

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PCT/US2016/044922 [0386] Arabinose inducible promoters are known in the art, including Para, ParaB, Parac, and ParaBAD· In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the Parac promoter and the ParaBAD promoter operate as a bidirectional promoter, with the ParaBAD promoter controlling expression of a heterologous gene(s) in one direction, and the Parac (in close proximity to, and on the opposite strand from the ParaBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

[0387] In one exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a Parac promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (Ρτ_εικ)· In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the engineered bacterial cell, and the engineered bacterial cell is killed. In one embodiment, the araC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.

[0388] In one embodiment of the disclosure, the engineered bacterial cell further comprises an antitoxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the antitoxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the engineered bacterial cell. The engineered bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the engineered bacterial cell will be killed by the toxin.

[0389] In another embodiment of the disclosure, the engineered bacterial cell further comprises an antitoxin under the control of the ParaBAD promoter. In this situation, in the -138WO 2017/023818

PCT/US2016/044922 presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the engineered bacterial cell. The engineered bacterial cell is no longer viable once the toxin protein is expressed, and the engineered bacterial cell will be killed by the toxin.

[0390] In another exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell (and required for survival), and a Parac promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the engineered bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and the essential protein required for survival is not expressed. In this case, the engineered bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the engineered bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anto-toxin killswitch system described directly above.

[0391] In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it.

[0392] In some embodiments, the engineered bacteria of the present disclosure, for example, bacteria described herein may further comprise the gene(s) encoding the components of any of the above-described kill-switch circuits.

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PCT/US2016/044922 [0393] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, lbs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin lb, colicin 5, colicinlO, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

[0394] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNAl, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccE^ctd, MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, ImlO, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

[0395] In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.

[0396] In one embodiment, the method further comprises administering a second engineered bacterial cell to the subject, wherein the second engineered bacterial cell comprises a heterologous reporter gene operably linked to an inducible promoter that is directly or indirectly induced by an exogenous environmental condition. In one embodiment, the heterologous reporter gene is a fluorescence gene. In one embodiment, the fluorescence gene encodes a green fluorescence protein (GFP). In another embodiment, the method further comprises administering a second engineered bacterial cell to the subject, wherein the second engineered bacterial cell expresses a lacZ reporter construct that cleaves a substrate to produce a small molecule that can be detected in urine (see, for example, Danio et al.,

Science Translational Medicine, 7(289):1-12, 2015, the entire contents of which are expressly incorporated herein by reference).

Isolated Plasmids [0397] In other embodiments, the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a first payload operably linked to a first inducible promoter, and

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PCT/US2016/044922 a second nucleic acid encoding a second payload operably linked to a second inducible promoter. In other embodiments, the disclosure provides an isolated plasmid further comprising a third nucleic acid encoding a third payload operably linked to a third inducible promoter. In other embodiments, the disclosure provides a plasmid comprising four, five, six, or more nucleic acids encoding four, five, six, or more payloads operably linked to inducible promoters. In any of the embodiments described here, the first, second, third, fourth, fifth, sixth, etc “payload(s)” can be a propionate catabolism enzyme, a propionate transporter, a propionate binding protein, or other sequence described herein. In one embodiment, the nucleic acid encoding the first payload and the nucleic acid encoding the second payload are operably linked to the first inducible promoter. In one embodiment, the nucleic acid encoding the first payload is operably linked to a first inducible promoter and the nucleic acid encoding the second payload is operably linked to a second inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In another embodiment, the first inducible promoter and the second inducible promoter are different inducible promoters. In other embodiments comprising a third nucleic acid, the nucleic acid encoding the third payload and the nucleic acid encoding the first and second payloads are all operably linked to the same inducible promoter. In other embodiments, the nucleic acid encoding the first payload is operably linked to a first inducible promoter, the nucleic acid encoding the second payload is operably linked to a second inducible promoter, and the nucleic acid encoding te third payload is operably linked to a third inducible promoter. In some embodiments, the first, second, and third inducible promoters are separate copies of the same inducible promoter. In other embodiments, the first inducible promoter, the second inducible promoter, and the third inducible promoter are different inducible promoters. In some embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter, are each directly or indirectly induced by low-oxygen or anaerobic conditions. In other embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter, are each a fumarate and nitrate reduction regulator (FNR) responsive promoter. In other embodiments, the first promoter, the second promoter, and the optional third promoter, or the first promoter and the second promoter and the optional third promoter are each a ROS-inducible regulatory region. In other embodiments, the first

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PCT/US2016/044922 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.

[0398] In some embodiments, the heterologous gene encoding a propionate catabolism enzyme is operably linked to a constitutive promoter. In one embodiment, the constitutive promoter is a lac promoter. In another embodiment, the constitutive promoter is a tet promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli σ32 promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli σ70 promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis σΑ promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis σΒ promoter. In another embodiment, the constitutive promoter is a Salmonella promoter. In other embodiments, the constitutive promoter is a bacteriophage T7 promoter. In other embodiments, the constitutive promoter is and a bacteriophage SP6 promoter. In any of the above-described embodiments, the plasmid further comprises a heterologous gene encoding a 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.

[0399] In some embodiments, 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. In another embodiment, 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.

[0400] In some embodiments, a first nucleic acid encoding a propionate catabolism enzyme comprises a prpE and/or a Pha gene. In other embodiments, a first nucleic acid encoding a propionate catabolism enzyme is a Pha gene or a Pha operon, e.g. prpE-phaBphaC-phaA. In some embodiments, 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. In other embodiments, a gene encoding a succinate -142WO 2017/023818

PCT/US2016/044922 exporter, e.g., SucEl and/or DcuC, is further expressed. In other embodiments, a priopionate importer is further expressed.

[0401] In some embodiments, a first nucleic acid encoding a propionate catabolism enzyme comprises a prpE and/or a MMCA pathway gene. In other embodiments, 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-accAl-pccB-mmcE-mutA-mutB or prpEaccAl-pccB or mmcE-mutA-mutB. In some embodiments, 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. In other embodiments, a gene encoding a succinate exporter, e.g., SucEl and/or DcuC, is further expressed. In other embodiments, a priopionate importer is further expressed.

[0402] In some embodiments, a first nucleic acid encoding a propionate catabolism enzyme comprises a prpE and/or a 2MC pathway gene. In other embodiments, 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. In some embodiments, the prpE and/or a 2MC pathway gene or a 2MC pathway operon is coexpressed with an additional propionate catabolism gene or gene cassette, e.g. a Pha cassette and/or a MMCA cassette described herein. In other embodiments, a gene encoding a succinate exporter, e.g., SucEl and/or DcuC, is further expressed. In other embodiments, a priopionate importer is further expressed.

[0403] In one embodiment, the plasmid is a high-copy plasmid. In another embodiment, the plasmid is a low-copy plasmid.

[0404] In another aspect, the disclosure provides a recombinant bacterial cell comprising an isolated plasmid described herein. In another embodiment, the disclosure provides a pharmaceutical composition comprising the recombinant bacterial cell.

[0405] In one embodiment, the bacterial cell further comprises a genetic mutation in an endogenous gene encoding a lysine acetyltransferase, e.g. pka, which propionylates and inactivates prpE. In another embodiment, the bacterial cell further comprises a genetic mutation which reduces export of propionate and/or its metabolites from the bacterial cell.

[0406] In one embodiment, 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.

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Multiple Mechanisms of Action [0407] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in FIG. 32. For example, 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. Alternatively, 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.

[0408] In some embodiments, 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) or gene cassette(s) encoding one or more antibiotic resistance(s), including but not limited to, kanamycin or chloramphenicol resistance; (7) one or more modifications that increase succinate export from the bacterial cell; (8) one or modifications that reduce succinate import into the bacterial cell; (9) mutations/deletions in genes, as described herein, e.g., pka, succinate importers or propionate exporters (10) mutations/deletions in genes of the endogenous propionate synthesis pathway.

[0409] In some embodiments, the genetically engineered bacteria comprise two or more different pathway cassettes or operons comprising propionate catabolism enzymes. In

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PCT/US2016/044922 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, AccAl, PccB, MmcE, MutA, and MutB, 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, accAl, 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. In some embodiments, 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-accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) in combination with one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA). In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accAlpccB-mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) in combination with one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpD-prpE). In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-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). In another non-limiting example of combinations, 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). In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpBprpC-prpD-prpE), and one or more MMCA pathway operon(s) (e.g., prpE-accAl-pccBmmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB).

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PCT/US2016/044922 [0410] Non limiting examples of combinations include genetically engineered bacteria comprising one or more MMCA pathway operon(s) (e.g., prpE-accAl-pccB-mmcEmutA-mutB, or prpE-accAl-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. In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accAlpccB-mmcE-mutA-mutB, or prpE-accAl-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. In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpEaccAl-pccB-mmcE-mutA-mutB, or prpE-accAl-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. In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more 2MC pathway operon(s) (e.g., prpB-prpC-prpDprpE), and one or more PHA pathway operon(s) (e.g., prpE-phaB-phaC-phaA) and in combination with one or more cassettes comprising MatB. In another non-limiting example of combinations, 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-accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-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.

[0411] In some embodiments, 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.

[0412] In some embodiments, 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. SucEl and/or dcuC. Non limiting examples of combinations include genetically engineered bacteria comprising one or more MMCA pathway operon(s) (e.g., prpE-accAl-pccB-mmcE-mutA-146WO 2017/023818

PCT/US2016/044922 mutB, or prpE-accAl-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. SucEl and/or dcuC. In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpE-accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) in combination with one or more 2MC pathway operon(s) (e.g., prpBprpC-prpD-prpE) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucEl and/or dcuC. In another non-limiting example of combinations, the genetically engineered bacteria comprise one or more MMCA pathway operon(s) (e.g., prpEaccAl-pccB-mmcE-mutA-mutB, or prpE-accAl-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. SucEl and/or dcuC. In another non-limiting example of combinations, 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., prpEphaB-phaC-phaA) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucEl and/or dcuC. In another non-limiting example of combinations, 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-accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) and one or more gene(s) or gene cassette(s) encoding one or more succinate exporters, e.g. SucEl and/or dcuC. In other non-limiting examples, 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. SucEl 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. In any of the embodiments, the engineered bacterium may also comprise gene sequence(s) encoding one or more propionate transporters.

[0413] In some embodiments, 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

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PCT/US2016/044922 propionate transporters. The engineered bacterium may further comprise gene sequence encoding one or more succinate exporters. Thus, in some embodiments 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. In some embodiments 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. In some embodiments 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.

[0414] In some embodiments, certain catalytic steps are rate limiting and in such a case it may be beneficial to add additional copies of one or more gene(s) encoding one or more rate limiting enzyme(s). In a non-limiting example, 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. In a non-limiting example, 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.

[0415] In a non-limiting example, the genetically engineered bacteria may encode one or more MMCA pathway operon(s) e.g., prpE-accAl-pccB-mmcE-mutA-mutB, or prpEaccAl-pccB and mmcE-mutA-mutB) and one or more additional gene(s) or gene cassette(s) encoding one or more of prpE and/or accAl and/or opccB and/or mmcE and/or mutA and/or mutB. In another non-limiting example, 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).

[0416] In some embodiments, each gene from a propionate catabolism pathway described herein, e.g., PHA, MMCA, and/or 2MC, can be expressed individually, each under control of a separate (same or different) promoter. For example, one or more of prpE and/or phaB and/or phaC and/or phaA can be expressed individually, each under control of a separate (same or different) promoter. For example, one or more of prpE and/or accAl and/or

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PCT/US2016/044922 opccB and/or mmcE and/or mutA and/or mutB can be expressed individually, each under control of a separate (same or different) promoter. For example, one or more of prpB and/or prpC and/or prpD and/or prpE can be expressed individually, each under control of a separate (same or different) promoter. In some embodiments, 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, AcclA, and PccB, (e.g., with PrpE)) can be expressed individually, each under control of a separate (same or different) promoter.

[0417] In certain embodiments the order of the genes within a gene cassette can be modified, e.g., to increase or decrease levels of a particular gene within a cassette. In a nonlimiting example, 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. In a non-limiting example, 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. In a non-limiting example, 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.

[0418] In a non-limiting example, 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. In a non-limiting example, the genetically engineered bacteria may encode one or more 2MC pathway operon(s) (e.g., prpBprpC-prpD-prpE), in which prpB comes second or prpC comes second or prpD comes second or prpE comes second. In a non-limiting example, 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. In a non-limiting example, 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.

[0419] In a non-limiting example, the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) in which prpE comes first or accAl comes first or pccB comes first or mmcE comes first or mutA comes first or mutB comes first. In a non-limiting example, the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-149WO 2017/023818

PCT/US2016/044922 accAl-pccB-mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) in which prpE comes second or accAl comes second or pccB comes second or mmcE comes second or mutA comes second or mutB comes second. In a non-limiting example, the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-accAl-pccB mmcE-mutA-mutB, or prpE-accAl-pccB and mmcE-mutA-mutB) in which prpE comes third or accAl comes third or pccB comes third or mmcE comes third or mutA comes third or mutB comes third. In a non-limiting example, the genetically engineered bacteria may encode one or more MMCA operon(s) (e.g., prpE-accAl-pccB-mmcE-mutA-mutB, or prpEaccAl-pccB and mmcE-mutA-mutB) in which prpE comes fourth, fifth or sixth or accAl 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. In some embodiments, matB comes first, second, third, fourth, fifith, or sixth in a gene cassette comprising matB.

[0420] In any of the embodiments described in this section or elsewhere in the specification, any one or more the genes can be operably linked to a 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.

[0421] In certain embodiments, ribosome binding sites, e.g., stronger or weaker ribosome binding sites can be used to modulate (increase or decrease) the levels of expression of a propionate catabolism enzyme within a cassette.

[0422] In some embodiments, the genetically engineered bacteria further comprise mutations or deletions, e.g., in pka, succinate importers or propionate exporters, and an auxo trophy.

Host-Plasmid Mutual Dependency [0423] In some embodiments, the genetically engineered bacteria also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans', (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broadspectrum toxin. The toxin gene may be used to select against plasmid spread by making the

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PCT/US2016/044922 plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wildtype bacterium). In some embodiments, the GeneGuard plasmid is stable for at least onehundred generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria described herein.

[0424] The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

[0425] In some embodiments, the vector comprises a conditional origin of replication. In some embodiments, the conditional origin of replication is a R6K or ColE2P9. In embodiments where the plasmid comprises the conditional origin of replication R6K, the host cell expresses the replication initiator protein π. In embodiments where the plasmid comprises the conditional origin or replication ColE2, the host cell expresses the replication initiator protein RepA. It is understood by those of skill in the art that the expression of the replication initiator protein may be regulated so that a desired expression level of the protein is achieved in the host cell to thereby control the replication of the plasmid. For example, in some embodiments, the expression of the gene encoding the replication initiator protein may be placed under the control of a strong, moderate, or weak promoter to regulate the expression of the protein.

[0426] In some embodiments, the vector comprises a gene encoding a protein required for complementation of a host cell auxotrophy, preferably a rich-media compatible auxotrophy. In some embodiments, the host cell is auxotrophic for thymidine (AthyA), and the vector comprises the thymidylate synthase (thyA) gene. In some embodiments, the host cell is auxotrophic for diaminopimelic acid (ΔάαρΑ) and the vector comprises the 4-hydroxytetrahydrodipicolinate 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

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PCT/US2016/044922 auxotrophy may be regulated so that a desired expression level of the protein is achieved in the host cell.

[0427] In some embodiments, the vector comprises a toxin gene. In some embodiments, the host cell comprises an anti-toxin gene encoding and/or required for the expression of an anti-toxin. In some embodiments, the toxin is Zeta and the anti-toxin is Epsilon. In some embodiments, the toxin is Kid, and the anti-toxin is Kis. In preferred embodiments, the toxin is bacteriostatic. Any of the toxin/antitoxin pairs described herein may be used in the vector systems of the present disclosure. It is understood by those of skill in the art that the expression of the gene encoding the toxin may be regulated using art known methods to prevent the expression levels of the toxin from being deleterious to a host cell that expresses the anti-toxin. For example, in some embodiments, the gene encoding the toxin may be regulated by a moderate promoter. In other embodiments, the gene encoding the toxin may be cloned adjacent to ribosomal binding site of interest to regulate the expression of the gene at desired levels (see, e.g., Wright et al. (2015)).

Integration [01] In some embodiments, any of the gene(s) or gene cassette(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. One or more copies of the heterologous gene or heterologous gene cassette may be integrated into the bacterial chromosome. Having multiple copies of the gene or gene cassette integrated into the chromosome allows for greater production of the corresponding protein(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the kill-switch circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

[02] For example, 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.

Secretion [0428] In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism (e.g., gram positive bacteria) or non-native secretion mechanism (e.g., gram negative bacteria) that is capable of secreting the propionate catabolism enzyme from the bacterial cytoplasm. Many bacteria have evolved sophisticated secretion systems to

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PCT/US2016/044922 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.

[0429] In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system. Double membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistancenodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in Figures

3-6. Mycobacteria, which have a Gram-negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane-spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).

[0430] In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type III-like secretion system (T3SS) from Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the propionate catabolism enzyme from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologouse protein or peptide, e.g., a propionate catabolism

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PCT/US2016/044922 enzyme, comprises a type III secretion sequence that allows the propionate catabolism enzyme to be secreted from the bacteria.

[0431] In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest, e.g., a propionate catabolism enzyme. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.

[0432] In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in Figure 10, a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex (‘Beta-barrel assembly machinery’) where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane-associated peptidase (black scissors; right side of Bam complex) to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologouse protein or peptide, e.g., a 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.

[0433] In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. 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 -154WO 2017/023818

PCT/US2016/044922 inner and outer membranes. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing Cterminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[0434] In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membranespanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in E. coli), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006;

Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the propionate catabolism enzymefrom the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.

[0435] In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g., therapeutic polypeptides) - particularly those of eukaryotic origin - contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

[0436] One way to secrete properly folded proteins in gram-negative bacteriaparticularly those requiring disulphide bonds - is to target the periplasm in bacteria with a -155WO 2017/023818

PCT/US2016/044922 destabilized outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These “leaky” gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a “leaky” or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nipt. Lpp is the most abundant polypeptide in the bacterial cell existing at -500,000 copies per cell and functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. 1.

Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are deactived. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpl can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[0437] To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected -156WO 2017/023818

PCT/US2016/044922 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). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., ove expression of colicins or the third topological domain of TolA, which peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

[0438] Table 9: The tables below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

Table 9. Secretion systems for gram positive bacteria

Bacterial Strain	Relevant Secretion System
C. novyi-NT (Gram+)	Sec pathway Twin- arginine (TAT) pathway
C. butryicum (Gram+)	Sec pathway Twin- arginine (TAT) pathway
Listeria monocytogenes (Gram +j	Sec pathway Twin- arginine (TAT) pathway

Table 10. Secretion Systems for Gram negative bacteria

Protein secretary pathways (SP) in gram-negative bacteria and their descendants

Type (Abbreviat ion)	Name	TC#²	Bact eria	Arch aea	Eukarya	# Protei ns/Sys tern	Energ y Sourc e
IMPS - Gram-negative bacterial inner membrane channel-forming translocases
ABC (SIP)	ATP binding cassette translocase	3.A.1	+	+	+	3-4	ATP

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SEC (IISP)	General secretory translocase	3.A.5	+	+	+	~12	GTP OR ATP + PMF
Fla/Path (IIISP)	Flagellum/vir ulence- related translocase	3.A.6	+			>10	ATP
Conj (IVSP)	Conjugation- related translocase	3.A.7	+			>10	ATP
Tat (IISP)	Twin- arginine targeting translocase	2.A.6 4	+	+	+ (chloroplas ts)	2-4	PMF
Oxal (YidC)	Cytochrome oxidase biogenesis family	2.A.9	+	+	+ (mitochon dria chloroplast s)	1	None or PMF
MscL	Large conductance mechanosens itive channel family	1.A.2 2	+	+	+	1	None
Holins	Holin functional superfamily	l.E.l •21	+			1	None
Eukaryotic Organelles
MPT	Mitochondria 1 protein translocase	3.A. B			+ (mitochon drial)	>20	ATP
CEPT	Chloroplast envelope protein translocase	3.A.9	(+)		+ (chloroplas ts)	>3	GTP
Bcl-2	Eukaryotic Bcl-2 family (programmed cell death)	1.A.2 1			+	1?	None
Gram-negative bacterial outer membrane channel-forming translocases
MTB (IISP)	Main terminal branch of the general secretory translocase	3.A.1 5				~14	ATP; PMF

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FUP AT-1	Fimbrial usher protein Autotransport er-1	l.B.l 1 l.B.l 2	f +^b	-	1 1	None None
AT-2 OMF (ISP)	Autotransport er-2	1.B.4 0 l.B.l 7	+^b +^b	+(?)	1 1	None None
TPS Secretin (IISP and IISP)		1.B.2 0 1.B.2 2	+ +^b	+	1 1	None None
OmpIP	Outer membrane insertion porin	1.B.3 3	+	+ (mitochon dria; chloroplast s)	>4	None ?

[0439] The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other propionate catabolism enzyme from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe / Volume 1, Number 9, 2006 “Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently”, the contents of which is herein incorporated by reference in its entirety.

[0440] In some embodiments, 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 susceptibility to proteases, stability, and/or half-life. In some embodiments, PrpE is secreted, alone or in combination other propionate catabolic enzymes, e.g.,with one or more of accAl, pccB, mmcE, mutA, and mutB and/or one or more of prpB, prpC, prpD, and/or one or more of phaB, phaC, phaA. In some embodiments, one or more of accAl, pccB, mmcE, mutA, mutB are secreted. In some embodiments, one or more of prpB, prpC, prpD are secreted. In some embodiments, one or more of phaB, phaC, phaA are secreted.

[0441] Alternatively, 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.

In Vivo Models [0442] 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

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PCT/US2016/044922 may be used. For example, 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 propionylcamitine and methyl citrate and dies within 36 hours of birth. However, the hypomorphic mouse of PCCA-/- (A138T) survives with elevated levels of propionic acidemia and hence it is a great model to use. Intravenous injections of adeno-associated virus 2/8 (AAV8) vectors to these hypomorphic mice reduced propionylcamitine and methyl citrate and mediated long lasting effects. A PCCA-/- knock-out mouse model can also be used (see, for example, Miyazaki et al., 2001, J. Biol. Chem., 276:35995-35999). A mouse model of Methylmalonic Acidemia has also been described by Peters et al. (see, for example, Peters et al., 2012, PLoS ONE, 7(7):e40609).

[0443] Alternatively, 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. For example, a serotype 9 rAAV expressing the Mut cDNA effectively rescued the Mut-/- mice from lethality, conferred long-term survival, markedly improved metabolism and resulted in striking preservation of renal function and histology (Senac et al., Gene therapy in a murine model of Methylmalonic Acidemia (MMA) using rAAV9 mediated gene delivery; Gene Ther. 2012 Apr; 19(4): 385-391). Another Mut (-/-) mouse has been described by Peters et al. (Peters et al., A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality; J Biol Chem. 2003 Dec 26;278(52):52909-13 and also Peters et al., 2012, PLoS ONE, 7(7):e40609).

[0444] 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 propionylcamitine, acetylcamitine, 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 propionylcamitine, acetylcamitine,

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PCT/US2016/044922 and/or methylcitrate after treatment indicates that the engineered bacteria are effective for treating the disease.

Methods of Screening

Generation of Bacterial Strains with Enhance Ability to Transport Metabolites or

Biomolecules [0445] Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

[0446] This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite or biomolecule.

[0447] For example, if the biosynthetic pathway for producing a certain metabolite or biomolecule is disrupted a strain capable of high-affinity capture of said metabolite or biomolecule can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid or metabolite, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the metabolite or biomolecule at regular intervals. Over time, cells that are most competitive for the metabolite or biomolecule - at growth-limiting concentrations - will come to dominate the population. These strains will likely have mutations in their metabolitetransporters resulting in increased ability to import the essential and limiting metabolite or biomolecule.

[0448] Similarly, by using an auxotroph that cannot use an upstream metabolite to form a certain metabolite or biomolecule, a strain can be evolved that not only can more efficiently imports the upstream metabolite, but also converts the metabolite into the essential

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PCT/US2016/044922 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.

[0449] A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound, this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate.

[0450] Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growthlimiting 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.

[0451] Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

[0452] Predicting when to halt the stop the ALE experiment also requires vigilance.

As the success of directing evolution is tied directly to the number of mutations “screened” throughout the experiment and mutations are generally a function of errors during DNA -162WO 2017/023818

PCT/US2016/044922 replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 1011.2 CCD1. This rate can be accelerated by the addition of chemical mutagens to the cultures - such as N-methyl-N-nitro-Nnitrosoguanidine (NTG) - which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

[0453] At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. 0. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

[0454] Similar methods can be used to generate E.Coli Nissle mutants that consume or import propionate and or one or more of its metabolites.

Pharmaceutical Compositions and Formulations [0455] Pharmaceutical 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.

[0456] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein, e.g., to express at least one propionate catabolism gene or gene cassette. In alternate embodiments, the pharmaceutical composition comprises two or

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PCT/US2016/044922 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).

[0457] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[0458] 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, delayedrelease, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10 to 10 bacteria, e.g., approximately 10 bacteria, approximately 10 bacteria, approximately 10 bacteria, approximately 10 bacteria, approximately 10⁹ bacteria, approximately 10¹⁰ bacteria, approximately 10¹¹ bacteria, or approximately 10¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly.

[0459] The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal.

[0460] 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 -164WO 2017/023818

PCT/US2016/044922 complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. The genetically engineered bacteria disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semisolid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the engineered bacteria may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.

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PCT/US2016/044922 [0461] 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.

[0462] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., /actose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (APMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,Ndimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-coglycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

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PCT/US2016/044922 [0463] In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

[0464] In some embodiments, enteric coating materials may be used, in one or more coating layers (e.g., outer, inner and/o intermediate coating layers). Enteric coated polymers remain unionised at low pH, and therefore remain insoluble. But as the pH increases in the gastrointestinal tract, the acidic functional groups are capable of ionisation, and the polymer swells or becomes soluble in the intestinal fluid.

[0465] 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 (HPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers. Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylose acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate.

[0466] Coating polymers also may comprise one or more of, phthalate derivatives,

CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, Eudragit™ S (poly(methacrylic acid, methyl methacrylate) 1:2);

Eudragit L100™ S (poly(methacrylic acid, methyl methacrylate)l:l); Eudragit L30D™, (poly(methacrylic acid, ethyl acrylate) 1:1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate) 1:1) (Eudragit™ 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, polyvinyl acetate 30D (30% dispersion in water), a neutral methacrylic ester comprising poly(dimethylaminoethylacrylate) (“Eudragit E™), a copolymer of methylmethacrylate and ethylacrylate with trimethylammonioethyl -167WO 2017/023818

PCT/US2016/044922 methacrylate chloride, a copolymer of methylmethacrylate and ethylacrylate, Zein, shellac, gums, or polysaccharides, or a combination thereof.

[0467] Coating layers may also include polymers which contain Hydroxypropylmethylcellulose (HPMC), Hydroxypropylethylcellulose (HPEC), Hydroxypropylcellulose (HPC), hydroxypropylethylcellulose (HPEC), hydroxymethylpropylcellulose (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), hydroxymethylethylcellulose (HMEC), propylhydroxyethylcellulose (PHEC), methylhydroxyethylcellulose (Μ H EC), hydrophobically modified hydroxyethylcellulose (NEXTON), carboxymethyl hydroxyethylcellulose (CMHEC), Methylcellulose, Ethylcellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates such as ammonia alginate, sodium alginate, potassium alginate, acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydroxypropylcellulose phthalate (HPCP), hydroxypropylethylcellulose phthalate (HPECP), hydroxyproplymethylcellulose phthalate (HPMCP), hydroxyproplymethylcellulose acetate succinate (HPMCAS).

[0468] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria described herein.

[0469] In one embodiment, the genetically engineered bacteria of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics,

134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric -168WO 2017/023818

PCT/US2016/044922 compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

[0470] In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

[0471] In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, flavor is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

[0472] In certain embodiments, the genetically engineered bacteria may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject’s diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[0473] In another embodiment, the pharmaceutical composition comprising the engineered bacteria may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional

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PCT/US2016/044922 food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the engineered bacteria are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the engineered bacteria are well known in the art. For example, see U.S. 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.

[0474] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[0475] The genetically engineered bacteria described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0476] 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

-170WO 2017/023818

PCT/US2016/044922 injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0477] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

[0478] 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.

[0479] In other embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and poly orthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[0480] 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 -171WO 2017/023818

PCT/US2016/044922 of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans. The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0481] The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).

Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%,

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PCT/US2016/044922 and poly sorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

Methods of Treatment [0482] 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. In one embodiment, 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 Bp deficiencies. In one embodiment, the disease associated with abnormal catabolism of propionate is propionic acidemia. In one embodiment, the disease associated with abnormal catabolism of propionate is methylmalonic acidemia. In another embodiment, the disease associated with abnormal catabolism of propionate is a vitamin Bp deficiency.

[0483] In one embodiment, 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, is an 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. Patients with PA are unable to properly process propionyl CoA, which can lead to the toxic accumulation of propionyl CoA and propionic 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).

[0484] Propionyl CoA Carboxylase (PCC) is a dodecameric enzyme comprised of alpha and beta subunits. The alpha subunit of PCC (also called PCCA; NM_000282) comprises the biotin carboxylase and biotin carboxyl carrier protein domains, while the beta

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PCT/US2016/044922 subunit (also called PCCB; NM_000532) contains the carboxyltransferase activity (Diacovich et al., Biochemistry, 43(44):14027-14036, 2004). Mutations in either the PPCA or PPCB genes can lead to the development of Propionic Acidemia, and more than twentyfour mutations in genes encoding PCCA or PCCB have been identified that result in 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.

[0485] In one embodiment, 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). However, over sixty percent of subjects with methylmalonic acidemia have mutations in the methylmalonyl CoA mutase (MUT) gene. MUT is responsible for converting methylmalonyl CoA into succinyl CoA and requires a vitamin B 12-derived prosthetic group, adenosylcoalamin (also known as AdoCbl) to function. Upon entry into the mitochondria, the mitochondrial leader sequence at the N-terminus of MUT is cleaved, and MUT monomers then associate into homodimers.

The methylmalonic aciduria type A protein, mitochondrial (also known as MMAA) aides AdoCbl loading onto MUT. Similarly, Cob(l)yrinic acid, a,c-diamind adenosyltransferase, mitochondrial (MMAB), is an enzyme that catalyzes the final step in the conversion of vitamin B12 into adenosylcobalamin (AdoCbl). Methylmalonic aciduria and homocystinura type C protein, mitochondrial (also known as MMACHC) and methylmalonic aciduria and homocystinurai type D protein, mitochondrial (also known as MMADHC) encode mitochondrial proteins that are also involved in vitamin Bi₂ (cobalamin) synthesis.

[0486] 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).

[0487] Because of the inability to properly breakdown amino acids completely, patients having a disease associated with catabolism of propionate accumulate different -174WO 2017/023818

PCT/US2016/044922 byproduct molecules in their blood and urine (Carrillo-Carrasco and Venditti, Gene Reviews. Seattle (WA): University of Washington, Seattle; 1993-2015). The abnormal levels of these by-product molecules are used as the main diagnostic criteria for diagnosing the disorder (See, e.g., Table 11).

Table 11. Breakdown Products of Propionate for Use as Biomarkers

Blood metabolite	LC-MS/MS method
Propionylcamitine	Yes
Methylcitrate	Yes
Glycine	Yes
Propionate	Yes (in vitro assay)
Urine metabolite	LC-MS/MS method
3 -hydroxypropionate	No
Methylcitrate	Yes
Tiglylglycine	No
Propionylglycine	No

[0488] Detectable urinary organic acids useful for diagnosis and markers include, but are not limited to, N-propionylglycine, N-tiglyglycine, 2-methyl-3-oxovaleric acid, 3hydroxy-2-methylbutyric acid, 2 methyl-3-oxobutyric acid, 3-hydroxy-n-valeric acid, 3-oxon-valeric acid [0489] Currently available treatments for Propionic Acidemia and Methylmalonic Acidemia are inadequate for the long term management of the disease and have severe limitations (Li et al., Liver Transplantation, 2015). A low protein diet, with micronutrient and vitamin supplementation, as necessary, is the widely accepted long-term disease management strategy for PA and MMA (Li et al., 2015). However, protein-intake restrictions can be particularly problematic and result in significant morbidity. Even with proper monitoring and patient compliance, protein dietary restrictions result in a high incidence of mental retardation and mortality (Li et al., 2015). Additional non-surgical chronic management regimens include L carnitine administration, antibiotics (metronidazole), Vit B12 for select MMA responsive patients (cblA>cblB>mut (-)), and amino acid dietary formulas (isoleucine/valine, glutamine, alanine supplementation), and

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PCT/US2016/044922 dialysis. Further, a few cases of PA and MM A have been treated by liver transplantation (Li et al., 2015), kidney transplantation or combined liver/kidney transplantation. However, the limited availability of donor organs, the costs associated with the transplantation itself, and the undesirable effects associated with continued immunosuppressant therapy limit the practicality of liver transplantation for treatment of disease. Therefore, there is significant unmet need for effective, reliable, and/or long-term treatment for PA and MMA.

[0490] The present disclosure surprisingly demonstrates that pharmaceutical compositions comprising the engineered bacterial cells may be used to treat metabolic diseases involving the abnormal catabolism of propionate, such as PA and MMA.

[0491] In one embodiment, 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.

[0492] In one embodiment, 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 MM AB 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.

[0493] In another aspect, 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. In one embodiment, the subject has a disease or disorder involving the catabolism of propionate. In one embodiment, the disorder involving the catabolism of propionate is a metabolic disorder involving the abnormal catabolism of propionate. In another embodiment, the disorder involving the catabolism of propionate is propionic acidemia. In another embodiment, the disorder involving the catabolism of propionate is methylmalonic acidemia. In another embodiment, the disorder involving the catabolism of propionate is a vitamin Bn deficiency.

[0494] In some embodiments, the disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to seizures, vomiting, lethargy, hypotonia, encephalopathy, developmental delay, failure to thrive, liver failure, and/or secondary hyperammonemia. In some embodiments, the disease is secondary to other conditions, e.g., liver disease.

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PCT/US2016/044922 [0495] In certain embodiments, 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. In some embodiments, the bacterial cells are delivered simultaneously with dietary protein. In another embodiment, the bacterial cells are delivered simultaneously with L-carnitine. In some embodiments, the bacterial cells and dietary protein are delivered after a period of fasting or protein-restricted dieting. In these embodiments, 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. In some embodiments, 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. In these embodiments, 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.

[0496] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria disclosed herein are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria are lyophilized in a gel cap and administered orally. In some embodiments, 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.

[0497] In certain embodiments, the pharmaceutical composition described herein is administered to reduce propionate, propionyl CoA, and/or methylmalonyl CoA levels in a subject. In some embodiments, 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. In another embodiment, 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-177WO 2017/023818

PCT/US2016/044922 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. In another embodiment, 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.

[0498] In one embodiment, 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.

[0499] In some embodiments, 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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, 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.

[0500] Before, during, and after the administration of the pharmaceutical composition, 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. In some embodiments, the methods may include administration of the compositions of the disclosure to reduce levels of the propionate, propionyl CoA, and/or methylmalonyl CoA. In some embodiments, 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 -178WO 2017/023818

PCT/US2016/044922 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.

[0501] In some embodiments, 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.

[0502] Certain unmodified bacteria will not have appreciable levels of propionyl CoA and/or methylmalonyl CoA processing. In embodiments using genetically modified forms of these bacteria, processing of propionyl CoA and/or methylmalonyl CoA will be appreciable under exogenous environmental conditions.

[0503] 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. In some embodiments, propionate catabolism enzyme, e.g., PrpBCDE, expression is measured by methods known in the art. In another embodiment, propionate catabolism enzyme activity is measured by methods known in the art to assess PrpBCDE activity (see propionate catabolism enzyme sections, supra). In another embodiment, propionate catabolism enzyme activity is measured by methods known in the art to assess activity of a PHA pathway circuit described herein. In another embodiment, propionate catabolism enzyme activity is measured by methods known in the art to assess the activity of a MMCA circuit described herein.

[0504] In certain embodiments, 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. Thus, 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. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.

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PCT/US2016/044922 [0505] In one embodiments, the bacterial cells are administered to a subject once daily. In another embodiment, the bacterial cells are administered to a subject twice daily. In another embodiment, the bacterial cells are administered to a subject three times daily. In another embodiment, the bacterial cells are administered to a subject in combination with a meal. In another embodiment, the bacterial cells are administered to a subject prior to a meal. In another embodiment, the bacterial cells are administered to a subject after a meal. 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.

[0506] 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-camitine, and/or a low-protein diet. The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents.

[0507] An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the bacteria, e.g., the agent(s) must not interfere with or kill the bacteria. In some embodiments, the pharmaceutical composition is administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food. The pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g., low-protein diet and amino acid supplementation. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.

[0508] 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 administration of a composition and determining the level of the propionate, propionyl CoA and/or methylmalonyl CoA in the sample.

[0509] In one embodiment, 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

-180WO 2017/023818

PCT/US2016/044922 subject before administration of a composition to the subject. In one embodiment, 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. In one embodiment, 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. In another embodiment, 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 administration of the pharmaceutical composition.

[0510] In one embodiment, 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.

[0511] 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 administration of a composition and determining the level of the propionate, propionyl CoA and/or methylmalonyl CoA in the sample.

[0512] In another embodiment, the methods further comprise comparing the level of methylcitrate, propionylcamitine, and/or acetylcamitine, and/or the propionylcamitine to acetylcamitine 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. In one embodiment, a reduced level of methylcitrate, propionylcamitine, and/or acetylcamitine the propionylcamitine to acetylcamitine ratio in the plasma sample from the subject after administration of a composition indicates that the plasma levels of methylcitrate, propionylcamitine, and/or acetylcamitine are decreased, thereby treating the disorder involving the catabolism of propionate in the subject. In one embodiment, the plasma level of methylcitrate, propionylcamitine, and/or acetylcamitine,

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PCT/US2016/044922 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 administration of the pharmaceutical composition. In another embodiment, 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.

[0513] In one embodiment, 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.

Examples [0514] The present disclosure is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents, and published patent applications, as cited throughout this application are hereby expressly incorporated herein by reference. It should further be understood that the contents of all the figures and tables attached hereto are also expressly incorporated herein by reference.

Development of Engineered Bacterial Cells

Example 1. Construction of Plasmids Encoding Propionate Catabolism Enzymes and Propionate Transporters (prpBCDE operon and mtC gene) [0515] Either the prpBCDE operon from E. coli strain Nissle (SEQ ID NO: 45) or Salmonella (SEQ ID NO: 94) are synthesized (Genewiz), fused to the Tet promoter, cloned into the high-copy plasmid pUC57-Kan by Gibson assembly, and transformed into E. coli DH5a as described herein to generate the plasmid pTet-prpBCDE. The mctC gene of Corynebacterium fused to the Tet promoter (SEQ ID NO: 88) is synthesized (Genewiz) and cloned into the high-copy plasmid pUC57-Kan to generate the plasmid pTet-mctC.

[0516] In certain constructs, the prpBCDE operon is operably linked to a FNRresponsive promoter, which may be is further fused to a strong ribosome binding site sequence. For efficient translation, each synthetic gene in the operon was separated by a 15

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PCT/US2016/044922 base pair ribosome binding site derived from the T7 promoter/translational start site. Each gene cassette and regulatory region construct is expressed on a high-copy plasmid, a lowcopy plasmid, or a chromosome.

[0517] In certain embodiments the construct is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., FIG. 32). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the propionate construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting E. coli Nissle bacteria are genetically engineered to express a propionate biosynthesis cassette and produce propionate.

Example 2. Construction of Plasmids Encoding Propionate Catabolism Enzymes (PHA Pathway) [0518] First, the E. coli Nissle prpE gene and phaBCA genes from Acinetobacter sp RA3849 were codon optimized for expression in E. coli Nissle, synthesized, and were placed under the control of an aTc-inducible promoter in a single operon in a high copy plasmid the ~10-copy plasmid pl5A-Kan by Golden Gate assembly, as shown in FIG. 10C and FIG. 11. Corresponding construct sequences are listed in Table 12.

Table 12. prpE-PhaBCA pathway circuit sequences

Description	Sequence	SEQ ID NO
Construct comprising TetR (reverse orientation, italic) and a prpE-PhaBCA gene cassette driven by a tet	Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaat aagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcgg catactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatac gcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaa ccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgt acctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttat tacgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatcta acatctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgta	SEQ ID NO:22

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PCT/US2016/044922 promoter (italic) (as shown in FIG. ii); ribosome binding sites are underlined; L3S2P11 terminator in italics and underline; his terminator in bold.

ggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattccgacctca ttaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcatTAA TTC

CACTCCCTATCAGTGATAGAGAAAAGTGAATAAGGCGTAA

GTTCAACAGGAGAGCATTATGTCTTTTAGCGAATTTTA

TCAGCGTTCGATTAACGAACCGGAGAAGTTCTGGGCC

GAGCAGGCCCGGCGTATTGACTGGCAGACGCCCTTTA

CGCAAACGCTCGACCACAGCAACCCGCCGTTTGCCCG

TTGGTTTTGTGAAGGCCGAACCAACTTGTGTCACAAC

GCTATCGACCGCTGGCTGGAGAAACAGCCAGAGGCGC

TGGCATTGATTGCCGTCTCTTCGGAAACAGAGGAAGA

GCGTACCTTTACCTTCCGCCAGTTACATGACGAAGTGA

ATGCGGTGGCGTCAATGCTGCGCTCACTGGGCGTGCA

GCGTGGCGATCGGGTGCTGGTGTATATGCCGATGATT

GCCGAAGCGCATATTACCCTGCTGGCCTGCGCGCGCA

TTGGTGCTATTCACTCGGTGGTGTTTGGGGGATTTGCT

TCGCACAGCGTGGCAACGCGAATTGATGACGCTAAAC

CGGTGCTGATTGTCTCGGCTGATGCCGGGGCGCGCGG

CGGTAAAATCATTCCGTATAAAAAATTGCTCGACGAT

GCGATAAGTCAGGCACAGCATCAGCCGCGTCACGTTT

TACTGGTGGATCGCGGGCTGGCGAAAATGGCGCGCGT

TAGCGGGCGGGATGTCGATTTCGCGTCGTTGCGCCAT

CAACACATCGGCGCGCGGGTGCCGGTGGCATGGCTGG

AATCCAACGAAACCTCCTGCATTCTCTACACCTCCGGC

ACGACCGGCAAACCTAAAGGTGTGCAGCGTGATGTCG

GCGGATATGCGGTGGCGCTGGCGACCTCGATGGACAC

CATTTTTGGCGGCAAAGCGGGCGGCGTGTTCTTTTGTG

CTTCGGATATCGGCTGGGTGGTAGGGCATTCGTATATC

GTTTACGCGCCGCTGCTGGCGGGGATGGCGACTATCG

TTTACGAAGGATTGCCGACCTGGCCGGACTGCGGCGT

GTGGTGGAAAATTGTCGAGAAATATCAGGTTAGCCGC

ATGTTCTCAGCGCCGACCGCCATTCGCGTGCTGAAAA

AATTCCCTACCGCTGAAATTCGCAAACACGATCTTTCG

TCGCTGGAAGTGCTCTATCTGGCTGGAGAACCGCTGG

ACGAGCCGACCGCCAGTTGGGTGAGCAATACGCTGGA

TGTGCCGGTCATCGACAACTACTGGCAGACCGAATCC

GGCTGGCCGATTATGGCGATTGCTCGCGGTCTGGATG

ACAGACCGACGCGTCTGGGAAGCCCCGGCGTGCCGAT

GTATGGCTATAACGTGCAGTTGCTCAATGAAGTCACC

GGCGAACCGTGTGGCGTCAATGAGAAAGGGATGCTGG

TAGTGGAGGGGCCATTGCCGCCAGGCTGTATTCAAAC

CATCTGGGGCGACGACGACCGCTTTGTGAAGACGTAC

TGGTCGCTGTTTTCCCGTCCGGTGTACGCCACTTTTGA

CTGGGGCATCCGCGATGCTGACGGTTATCACTTTATTC

TCGGGCGCACTGACGATGTGATTAACGTTGCCGGACA

TCGGCTGGGTACGCGTGAGATTGAAGAGAGTATCTCC

AGTCATCCGGGCGTTGCCGAAGTGGCGGTGGTTGGGG

TGAAAGATGCGCTGAAAGGGCAGGTGGCGGTGGCGTT

TGTCATTCCGAAAGAGAGCGACAGTCTGGAAGACCGT

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GAGGTGGCGCACTCGCAAGAGAAGGCGATTATGGCGC

TGGTGGACAGCCAGATTGGCAACTTTGGCCGCCCGGC

GCACGTCTGGTTTGTCTCGCAATTGCCAAAAACGCGA

TCCGGAAAAATGCTGCGCCGCACGATCCAGGCGATTT

GCGAAGGACGCGATCCTGGGGATCTGACGACCATTGA

TGATCCGGCGTCGTTGGATCAGATCCGCCAGGCGATG

GAAGAGTAGTACTGATCAAAAAGGTTAGCCTCAAGAG

GGTCATAAAAATGTCAGAGCAGAAAGTAGCTCTGGTT

ACCGGTGCGTTAGGTGGTATCGGAAGTGAGATCTGCC

GCCAGCTTGTGACCGCCGGGTACAAGATTATCGCCAC

CGTTGTTCCACGCGAAGAAGACCGCGAAAAACAATGG

TTGCAAAGTGAGGGGTTTCAAGACTCTGATGTGCGTTT

CGTATTAACAGATTTAAACAATCACGAAGCTGCGACA

GCGGCAATTCAAGAAGCGATTGCCGCCGAAGGACGCG

TTGATGTATTGGTCAACAACGCGGGGATCACGCGCGA

TGCTACATTTAAGAAAATGTCCTATGAGCAATGGTCC

CAAGTCATCGACACGAATTTAAAGACTCTTTTTACCGT

GACCCAGCCAGTATTTAATAAAATGCTTGAACAGAAG

TCTGGCCGCATCGTAAACATTAGCTCTGTCAATGGTTT

AAAAGGGCAATTTGGTCAAGCCAACTACTCGGCCTCG

AAAGCAGGGATTATCGGGTTTACTAAAGCATTGGCGC

AGGAGGGTGCTCGCTCGAACATTTGCGTCAATGTCGT

TGCTCCTGGTTACACAGCGACACCCATGGTCACAGCA

ATGCGCGAGGATGTAATTAAGTCAATCGAAGCTCAAA

TTCCCCTGCAACGTCTGGCAGCACCGGCGGAGATTGC

GGCAGCGGTTATGTATTTGGTGAGTGAACACGGTGCA

TACGTGACGGGCGAAACTTTGAGTATCAACGGCGGGC

TGTACATGCACTAAAGGTGCTTTTAGTCTAGCGCTAGA

GCAGGTACCATATTAATGAATCCAAATTCCTTTCAGTT

TAAAGAGAATATCTTACAGTTTTTCAGCGTGCACGAC

GATATTTGGAAAAAACTGCAGGAATTTTACTATGGAC

AATCGCCCATCAATGAAGCGTTGGCGCAGTTAAATAA

GGAAGACATGAGTTTATTCTTCGAGGCGTTATCAAAA

AACCCTGCTCGTATGATGGAGATGCAGTGGTCCTGGT

GGCAAGGGCAGATTCAAATTTACCAGAACGTGTTAAT

GCGTAGTGTAGCCAAGGACGTAGCCCCCTTTATCCAG

CCAGAGTCCGGAGATCGTCGCTTCAACTCGCCACTTTG

GCAAGAACATCCAAATTTTGATTTACTGAGTCAATCCT

ACTTGTTGTTTTCTCAGTTGGTTCAAAATATGGTGGAT

GTCGTTGAAGGAGTACCTGATAAGGTCCGCTATCGCA

TCCATTTCTTTACACGTCAGATGATCAATGCGTTGTCT

CCTTCTAATTTCCTGTGGACGAACCCTGAAGTAATTCA

ACAGACGGTCGCTGAACAGGGTGAGAATTTAGTACGC

GGGATGCAAGTATTTCACGATGATGTAATGAATTCGG

GTAAATATTTGAGCATCCGTATGGTAAATAGCGACAG

TTTCTCTCTTGGCAAGGACTTGGCGTATACGCCAGGAG

CCGTAGTTTTCGAGAACGACATCTTTCAGCTTCTTCAA

TACGAAGCCACAACCGAGAACGTATATCAAACCCCTA

TTCTTGTCGTACCTCCCTTCATCAACAAGTACTACGTG

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	CTGGACCTGCGCGAACAGAATAGCTTGGTTAATTGGC TGCGCCAACAAGGACATACGGTGTTTTTGATGTCGTG GCGTAACCCCAACGCAGAGCAGAAGGAGCTTACCTTC GCTGACTTAATTACCCAAGGATCGGTAGAAGCATTAC GTGTTATCGAAGAAATCACGGGAGAGAAAGAAGCTA ACTGTATTGGATATTGCATCGGTGGTACACTTCTGGCT GCTACCCAGGCATATTATGTAGCTAAACGCCTGAAAA ATCACGTAAAGTCAGCGACTTATATGGCGACGATTAT TGATTTTGAGAACCCCGGCTCATTGGGTGTTTTCATTA ATGAGCCGGTCGTAAGTGGACTTGAAAACCTTAATAA TCAACTTGGTTACTTCGACGGGCGTCAACTTGCAGTGA CATTTTCGTTGTTGCGCGAAAACACCTTGTATTGGAAT TATTACATCGATAATTACTTGAAGGGTAAGGAACCGT CCGACTTTGACATCTTATACTGGAACTCGGATGGTACG AATATCCCAGCAAAGATTCACAATTTCCTGTTACGTAA CCTTTATCTTAACAACGAACTTATTTCTCCAAATGCCG TCAAAGTTAATGGTGTGGGTTTAAACCTTTCGCGCGTG AAGACTCCATCATTCTTCATTGCTACGCAGGAGGACC ATATCGCATTGTGGGATACCTGTTTTCGCGGCGCGGAT TACCTGGGGGGTGAGAGCACACTTGTGCTTGGGGAAA GCGGACACGTCGCCGGCATTGTCAACCCGCCTTCTCGT AACAAGTATGGTTGTTACACGAACGCCGCCAAGTTTG AAAATACCAAGCAATGGCTTGACGGTGCAGAATATCA TCCCGAAAGCTGGTGGTTACGTTGGCAGGCATGGGTC ACGCCTTATACTGGAGAGCAGGTTCCTGCGCGTAATTT GGGAAACGCACAGTACCCCAGTATTGAAGCGGCCCCT GGGCGTTATGTGCTGGTAAACCTGTTTTAACGCTCACA TACAAGCAATCTATAATTATTCACGGTATAAATGAAA
GATGTTGTTATCGTAGCCGCTAAACGCACTGCGATCG GTTCCTTTCTGGGGAGTCTGGCTTCCCTGAGCGCCCCT CAGTTGGGTCAGACGGCTATCCGCGCAGTTTTGGATTC TGCAAATGTGAAACCAGAACAAGTGGACCAAGTAATT ATGGGGAATGTGCTGACCACCGGCGTTGGGCAAAATC CTGCTCGTCAGGCAGCAATCGCCGCTGGGATTCCTGT ACAAGTTCCCGCCAGCACGCTTAATGTAGTGTGTGGG TCCGGATTACGTGCCGTTCACCTGGCAGCTCAAGCCAT CCAATGCGATGAAGCCGATATCGTCGTTGCCGGAGGT CAAGAATCAATGTCCCAGTCTGCTCATTACATGCAGCT TCGCAATGGCCAGAAAATGGGTAACGCACAGTTAGTC GATTCAATGGTGGCCGACGGCTTGACCGACGCGTATA ATCAATACCAGATGGGTATCACCGCGGAGAATATCGT CGAAAAACTTGGTCTTAATCGTGAAGAACAAGACCAG CTTGCTCTGACAAGTCAACAACGTGCTGCAGCAGCGC AGGCTGCCGGAAAATTCAAGGATGAAATTGCGGTCGT TTCGATTCCCCAGCGCAAAGGAGAGCCGGTCGTCTTC GCGGAAGACGAATATATCAAGGCCAATACCTCGTTGG AATCCTTGACGAAACTGCGTCCAGCATTCAAAAAAGA CGGTTCTGTTACAGCCGGCAACGCATCTGGCATTAAT GATGGGGCAGCCGCGGTCCTGATGATGTCCGCCGACA

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	AAGCGGCTGAACTGGGCTTAAAGCCTTTAGCACGCAT TAAAGGTTACGCGATGTCAGGAATTGAGCCGGAAATC ATGGGACTGGGTCCTGTAGACGCCGTTAAGAAAACCC TTAATAAGGCTGGTTGGTCCTTAGACCAGGTCGATCTG ATCGAGGCCAATGAGGCTTTTGCTGCCCAAGCACTGG GAGTAGCCAAGGAGCTTGGGCTGGACCTGGACAAGGT AAATGTTAACGGAGGTGCGATCGCGCTGGGACACCCG ATCGGGGCTTCGGGTTGTCGTATCTTGGTCACGTTATT ACACGAAATGCAGCGTCGTGATGCAAAGAAGGGTATC GCCACATTGTGTGTGGGAGGTGGAATGGGGGTGGCGC TTGCCGTTGAGCGCGATTAAGGAGGTCGGATAAGGCG CTCGCGCCGCATCCGACACCGTGCGCAGATGCCTGAT GCGACGCTGACGCGTCTTATCATGCCTCGCTCTCGAGT CCCGTCAAGTCAGACGATCGCACGCCCCATGTGAACG ATTGGTAAACCCGGTGAACGCATGAGAAAGCCCCCG GAAGATCACCTTCCGGGGGCTTTTTTATTGCGCGG ACCAAAACGAAAAAAGACGCTCGAAAGCGTCTCTTTTCTG
GAA77TGG7ACCGAGGCGTAATGCTCTGCCAGTGTTAC AACCAATTAACCAATTCTGAT
Construct comprising a prpE-PhaBCA gene cassette under the control of the Ptet promoter(italic ) (as shown in FIG. 11) ribosome binding sites are underlined ;.L3S2P11 terminator in italics and underline; his terminator in bold	TTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAATAAG GCGTAAGGCGTAAGTTCAACAGGAGAGCATTATGTCT TTTAGCGAATTTTATCAGCGTTCGATTAACGAACCGGA GAAGTTCTGGGCCGAGCAGGCCCGGCGTATTGACTGG CAGACGCCCTTTACGCAAACGCTCGACCACAGCAACC CGCCGTTTGCCCGTTGGTTTTGTGAAGGCCGAACCAAC TTGTGTCACAACGCTATCGACCGCTGGCTGGAGAAAC AGCCAGAGGCGCTGGCATTGATTGCCGTCTCTTCGGA AACAGAGGAAGAGCGTACCTTTACCTTCCGCCAGTTA CATGACGAAGTGAATGCGGTGGCGTCAATGCTGCGCT CACTGGGCGTGCAGCGTGGCGATCGGGTGCTGGTGTA TATGCCGATGATTGCCGAAGCGCATATTACCCTGCTG GCCTGCGCGCGCATTGGTGCTATTCACTCGGTGGTGTT TGGGGGATTTGCTTCGCACAGCGTGGCAACGCGAATT GATGACGCTAAACCGGTGCTGATTGTCTCGGCTGATG CCGGGGCGCGCGGCGGTAAAATCATTCCGTATAAAAA ATTGCTCGACGATGCGATAAGTCAGGCACAGCATCAG CCGCGTCACGTTTTACTGGTGGATCGCGGGCTGGCGA AAATGGCGCGCGTTAGCGGGCGGGATGTCGATTTCGC GTCGTTGCGCCATCAACACATCGGCGCGCGGGTGCCG GTGGCATGGCTGGAATCCAACGAAACCTCCTGCATTC TCTACACCTCCGGCACGACCGGCAAACCTAAAGGTGT GCAGCGTGATGTCGGCGGATATGCGGTGGCGCTGGCG ACCTCGATGGACACCATTTTTGGCGGCAAAGCGGGCG GCGTGTTCTTTTGTGCTTCGGATATCGGCTGGGTGGTA GGGCATTCGTATATCGTTTACGCGCCGCTGCTGGCGG GGATGGCGACTATCGTTTACGAAGGATTGCCGACCTG GCCGGACTGCGGCGTGTGGTGGAAAATTGTCGAGAAA	SEQ ID NO: 23

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TATCAGGTTAGCCGCATGTTCTCAGCGCCGACCGCCAT

TCGCGTGCTGAAAAAATTCCCTACCGCTGAAATTCGC

AAACACGATCTTTCGTCGCTGGAAGTGCTCTATCTGGC

TGGAGAACCGCTGGACGAGCCGACCGCCAGTTGGGTG

AGCAATACGCTGGATGTGCCGGTCATCGACAACTACT

GGCAGACCGAATCCGGCTGGCCGATTATGGCGATTGC

TCGCGGTCTGGATGACAGACCGACGCGTCTGGGAAGC

CCCGGCGTGCCGATGTATGGCTATAACGTGCAGTTGC

TCAATGAAGTCACCGGCGAACCGTGTGGCGTCAATGA

GAAAGGGATGCTGGTAGTGGAGGGGCCATTGCCGCCA

GGCTGTATTCAAACCATCTGGGGCGACGACGACCGCT

TTGTGAAGACGTACTGGTCGCTGTTTTCCCGTCCGGTG

TACGCCACTTTTGACTGGGGCATCCGCGATGCTGACG

GTTATCACTTTATTCTCGGGCGCACTGACGATGTGATT

AACGTTGCCGGACATCGGCTGGGTACGCGTGAGATTG

AAGAGAGTATCTCCAGTCATCCGGGCGTTGCCGAAGT

GGCGGTGGTTGGGGTGAAAGATGCGCTGAAAGGGCA

GGTGGCGGTGGCGTTTGTCATTCCGAAAGAGAGCGAC

AGTCTGGAAGACCGTGAGGTGGCGCACTCGCAAGAGA

AGGCGATTATGGCGCTGGTGGACAGCCAGATTGGCAA

CTTTGGCCGCCCGGCGCACGTCTGGTTTGTCTCGCAAT

TGCCAAAAACGCGATCCGGAAAAATGCTGCGCCGCAC

GATCCAGGCGATTTGCGAAGGACGCGATCCTGGGGAT

CTGACGACCATTGATGATCCGGCGTCGTTGGATCAGA

TCCGCCAGGCGATGGAAGAGTAGTACTGATCAAAAAG

GTTAGCCTCAAGAGGGTCATAAAAATGTCAGAGCAGA

AAGTAGCTCTGGTTACCGGTGCGTTAGGTGGTATCGG

AAGTGAGATCTGCCGCCAGCTTGTGACCGCCGGGTAC

AAGATTATCGCCACCGTTGTTCCACGCGAAGAAGACC

GCGAAAAACAATGGTTGCAAAGTGAGGGGTTTCAAGA

CTCTGATGTGCGTTTCGTATTAACAGATTTAAACAATC

ACGAAGCTGCGACAGCGGCAATTCAAGAAGCGATTGC

CGCCGAAGGACGCGTTGATGTATTGGTCAACAACGCG

GGGATCACGCGCGATGCTACATTTAAGAAAATGTCCT

ATGAGCAATGGTCCCAAGTCATCGACACGAATTTAAA

GACTCTTTTTACCGTGACCCAGCCAGTATTTAATAAAA

TGCTTGAACAGAAGTCTGGCCGCATCGTAAACATTAG

CTCTGTCAATGGTTTAAAAGGGCAATTTGGTCAAGCC

AACTACTCGGCCTCGAAAGCAGGGATTATCGGGTTTA

CTAAAGCATTGGCGCAGGAGGGTGCTCGCTCGAACAT

TTGCGTCAATGTCGTTGCTCCTGGTTACACAGCGACAC

CCATGGTCACAGCAATGCGCGAGGATGTAATTAAGTC

AATCGAAGCTCAAATTCCCCTGCAACGTCTGGCAGCA

CCGGCGGAGATTGCGGCAGCGGTTATGTATTTGGTGA

GTGAACACGGTGCATACGTGACGGGCGAAACTTTGAG

TATCAACGGCGGGCTGTACATGCACTAAAGGTGCTTT

TAGTCTAGCGCTAGAGCAGGTACCATATTAATGAATC

CAAATTCCTTTCAGTTTAAAGAGAATATCTTACAGTTT

TTCAGCGTGCACGACGATATTTGGAAAAAACTGCAGG

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AATTTTACTATGGACAATCGCCCATCAATGAAGCGTT

GGCGCAGTTAAATAAGGAAGACATGAGTTTATTCTTC

GAGGCGTTATCAAAAAACCCTGCTCGTATGATGGAGA

TGCAGTGGTCCTGGTGGCAAGGGCAGATTCAAATTTA

CCAGAACGTGTTAATGCGTAGTGTAGCCAAGGACGTA

GCCCCCTTTATCCAGCCAGAGTCCGGAGATCGTCGCTT

CAACTCGCCACTTTGGCAAGAACATCCAAATTTTGATT

TACTGAGTCAATCCTACTTGTTGTTTTCTCAGTTGGTTC

AAAATATGGTGGATGTCGTTGAAGGAGTACCTGATAA

GGTCCGCTATCGCATCCATTTCTTTACACGTCAGATGA

TCAATGCGTTGTCTCCTTCTAATTTCCTGTGGACGAAC

CCTGAAGTAATTCAACAGACGGTCGCTGAACAGGGTG

AGAATTTAGTACGCGGGATGCAAGTATTTCACGATGA

TGTAATGAATTCGGGTAAATATTTGAGCATCCGTATG

GTAAATAGCGACAGTTTCTCTCTTGGCAAGGACTTGG

CGTATACGCCAGGAGCCGTAGTTTTCGAGAACGACAT

CTTTCAGCTTCTTCAATACGAAGCCACAACCGAGAAC

GTATATCAAACCCCTATTCTTGTCGTACCTCCCTTCAT

CAACAAGTACTACGTGCTGGACCTGCGCGAACAGAAT

AGCTTGGTTAATTGGCTGCGCCAACAAGGACATACGG

TGTTTTTGATGTCGTGGCGTAACCCCAACGCAGAGCA

GAAGGAGCTTACCTTCGCTGACTTAATTACCCAAGGA

TCGGTAGAAGCATTACGTGTTATCGAAGAAATCACGG

GAGAGAAAGAAGCTAACTGTATTGGATATTGCATCGG

TGGTACACTTCTGGCTGCTACCCAGGCATATTATGTAG

CTAAACGCCTGAAAAATCACGTAAAGTCAGCGACTTA

TATGGCGACGATTATTGATTTTGAGAACCCCGGCTCAT

TGGGTGTTTTCATTAATGAGCCGGTCGTAAGTGGACTT

GAAAACCTTAATAATCAACTTGGTTACTTCGACGGGC

GTCAACTTGCAGTGACATTTTCGTTGTTGCGCGAAAAC

ACCTTGTATTGGAATTATTACATCGATAATTACTTGAA

GGGTAAGGAACCGTCCGACTTTGACATCTTATACTGG

AACTCGGATGGTACGAATATCCCAGCAAAGATTCACA

ATTTCCTGTTACGTAACCTTTATCTTAACAACGAACTT

ATTTCTCCAAATGCCGTCAAAGTTAATGGTGTGGGTTT

AAACCTTTCGCGCGTGAAGACTCCATCATTCTTCATTG

CTACGCAGGAGGACCATATCGCATTGTGGGATACCTG

TTTTCGCGGCGCGGATTACCTGGGGGGTGAGAGCACA

CTTGTGCTTGGGGAAAGCGGACACGTCGCCGGCATTG

TCAACCCGCCTTCTCGTAACAAGTATGGTTGTTACACG

AACGCCGCCAAGTTTGAAAATACCAAGCAATGGCTTG

ACGGTGCAGAATATCATCCCGAAAGCTGGTGGTTACG

TTGGCAGGCATGGGTCACGCCTTATACTGGAGAGCAG

GTTCCTGCGCGTAATTTGGGAAACGCACAGTACCCCA

GTATTGAAGCGGCCCCTGGGCGTTATGTGCTGGTAAA

CCTGTTTTAACGCTCACATACAAGCAATCTATAATTAT

TCACGGTATAAATGAAAGATGTTGTTATCGTAGCCGC

TAAACGCACTGCGATCGGTTCCTTTCTGGGGAGTCTGG

CTTCCCTGAGCGCCCCTCAGTTGGGTCAGACGGCTATC

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	CGCGCAGTTTTGGATTCTGCAAATGTGAAACCAGAAC AAGTGGACCAAGTAATTATGGGGAATGTGCTGACCAC CGGCGTTGGGCAAAATCCTGCTCGTCAGGCAGCAATC GCCGCTGGGATTCCTGTACAAGTTCCCGCCAGCACGC TTAATGTAGTGTGTGGGTCCGGATTACGTGCCGTTCAC CTGGCAGCTCAAGCCATCCAATGCGATGAAGCCGATA TCGTCGTTGCCGGAGGTCAAGAATCAATGTCCCAGTC TGCTCATTACATGCAGCTTCGCAATGGCCAGAAAATG GGTAACGCACAGTTAGTCGATTCAATGGTGGCCGACG GCTTGACCGACGCGTATAATCAATACCAGATGGGTAT CACCGCGGAGAATATCGTCGAAAAACTTGGTCTTAAT CGTGAAGAACAAGACCAGCTTGCTCTGACAAGTCAAC AACGTGCTGCAGCAGCGCAGGCTGCCGGAAAATTCAA GGATGAAATTGCGGTCGTTTCGATTCCCCAGCGCAAA GGAGAGCCGGTCGTCTTCGCGGAAGACGAATATATCA AGGCCAATACCTCGTTGGAATCCTTGACGAAACTGCG TCCAGCATTCAAAAAAGACGGTTCTGTTACAGCCGGC AACGCATCTGGCATTAATGATGGGGCAGCCGCGGTCC TGATGATGTCCGCCGACAAAGCGGCTGAACTGGGCTT AAAGCCTTTAGCACGCATTAAAGGTTACGCGATGTCA GGAATTGAGCCGGAAATCATGGGACTGGGTCCTGTAG ACGCCGTTAAGAAAACCCTTAATAAGGCTGGTTGGTC CTTAGACCAGGTCGATCTGATCGAGGCCAATGAGGCT TTTGCTGCCCAAGCACTGGGAGTAGCCAAGGAGCTTG GGCTGGACCTGGACAAGGTAAATGTTAACGGAGGTGC GATCGCGCTGGGACACCCGATCGGGGCTTCGGGTTGT CGTATCTTGGTCACGTTATTACACGAAATGCAGCGTCG TGATGCAAAGAAGGGTATCGCCACATTGTGTGTGGGA GGTGGAATGGGGGTGGCGCTTGCCGTTGAGCGCGATT AAGGAGGTCGGATAAGGCGCTCGCGCCGCATCCGACA CCGTGCGCAGATGCCTGATGCGACGCTGACGCGTCTT ATCATGCCTCGCTCTCGAGTCCCGTCAAGTCAGACGAT CGCACGCCCCATGTGAACGATTGGTAAACCCGGTGAA CGCATGAGAAAGCCCCCGGAAGATCACCTTCCGGG GGCTTTTTTATTGCGCGGACCAAAACGAAAAAAGACGC
TCGAAAGCGTCTCTTTTCTGGAATTTGGTACCGAGGCGTN
ATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGA T
Construct comprising a prpE-PhaBCA gene cassette; (as shown in FIG. 11) ribosome binding sites are underlined	TAAGGCGTAAGTTCAACAGGAGAGCATTATGTCTTTT	SEQ ID NO: 24
AGCGAATTTTATCAGCGTTCGATTAACGAACCGGAGA AGTTCTGGGCCGAGCAGGCCCGGCGTATTGACTGGCA GACGCCCTTTACGCAAACGCTCGACCACAGCAACCCG CCGTTTGCCCGTTGGTTTTGTGAAGGCCGAACCAACTT GTGTCACAACGCTATCGACCGCTGGCTGGAGAAACAG CCAGAGGCGCTGGCATTGATTGCCGTCTCTTCGGAAA CAGAGGAAGAGCGTACCTTTACCTTCCGCCAGTTACA TGACGAAGTGAATGCGGTGGCGTCAATGCTGCGCTCA CTGGGCGTGCAGCGTGGCGATCGGGTGCTGGTGTATA

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TGCCGATGATTGCCGAAGCGCATATTACCCTGCTGGC

CTGCGCGCGCATTGGTGCTATTCACTCGGTGGTGTTTG

GGGGATTTGCTTCGCACAGCGTGGCAACGCGAATTGA

TGACGCTAAACCGGTGCTGATTGTCTCGGCTGATGCC

GGGGCGCGCGGCGGTAAAATCATTCCGTATAAAAAAT

TGCTCGACGATGCGATAAGTCAGGCACAGCATCAGCC

GCGTCACGTTTTACTGGTGGATCGCGGGCTGGCGAAA

ATGGCGCGCGTTAGCGGGCGGGATGTCGATTTCGCGT

CGTTGCGCCATCAACACATCGGCGCGCGGGTGCCGGT

GGCATGGCTGGAATCCAACGAAACCTCCTGCATTCTC

TACACCTCCGGCACGACCGGCAAACCTAAAGGTGTGC

AGCGTGATGTCGGCGGATATGCGGTGGCGCTGGCGAC

CTCGATGGACACCATTTTTGGCGGCAAAGCGGGCGGC

GTGTTCTTTTGTGCTTCGGATATCGGCTGGGTGGTAGG

GCATTCGTATATCGTTTACGCGCCGCTGCTGGCGGGG

ATGGCGACTATCGTTTACGAAGGATTGCCGACCTGGC

CGGACTGCGGCGTGTGGTGGAAAATTGTCGAGAAATA

TCAGGTTAGCCGCATGTTCTCAGCGCCGACCGCCATTC

GCGTGCTGAAAAAATTCCCTACCGCTGAAATTCGCAA

ACACGATCTTTCGTCGCTGGAAGTGCTCTATCTGGCTG

GAGAACCGCTGGACGAGCCGACCGCCAGTTGGGTGAG

CAATACGCTGGATGTGCCGGTCATCGACAACTACTGG

CAGACCGAATCCGGCTGGCCGATTATGGCGATTGCTC

GCGGTCTGGATGACAGACCGACGCGTCTGGGAAGCCC

CGGCGTGCCGATGTATGGCTATAACGTGCAGTTGCTC

AATGAAGTCACCGGCGAACCGTGTGGCGTCAATGAGA

AAGGGATGCTGGTAGTGGAGGGGCCATTGCCGCCAGG

CTGTATTCAAACCATCTGGGGCGACGACGACCGCTTT

GTGAAGACGTACTGGTCGCTGTTTTCCCGTCCGGTGTA

CGCCACTTTTGACTGGGGCATCCGCGATGCTGACGGTT

ATCACTTTATTCTCGGGCGCACTGACGATGTGATTAAC

GTTGCCGGACATCGGCTGGGTACGCGTGAGATTGAAG

AGAGTATCTCCAGTCATCCGGGCGTTGCCGAAGTGGC

GGTGGTTGGGGTGAAAGATGCGCTGAAAGGGCAGGT

GGCGGTGGCGTTTGTCATTCCGAAAGAGAGCGACAGT

CTGGAAGACCGTGAGGTGGCGCACTCGCAAGAGAAG

GCGATTATGGCGCTGGTGGACAGCCAGATTGGCAACT

TTGGCCGCCCGGCGCACGTCTGGTTTGTCTCGCAATTG

CCAAAAACGCGATCCGGAAAAATGCTGCGCCGCACGA

TCCAGGCGATTTGCGAAGGACGCGATCCTGGGGATCT

GACGACCATTGATGATCCGGCGTCGTTGGATCAGATC

CGCCAGGCGATGGAAGAGTAGTACTGATCAAAAAGGT

TAGCCTCAAGAGGGTCATAAAAATGTCAGAGCAGAAA

GTAGCTCTGGTTACCGGTGCGTTAGGTGGTATCGGAA

GTGAGATCTGCCGCCAGCTTGTGACCGCCGGGTACAA

GATTATCGCCACCGTTGTTCCACGCGAAGAAGACCGC

GAAAAACAATGGTTGCAAAGTGAGGGGTTTCAAGACT

CTGATGTGCGTTTCGTATTAACAGATTTAAACAATCAC

GAAGCTGCGACAGCGGCAATTCAAGAAGCGATTGCCG

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	CCGAAGGACGCGTTGATGTATTGGTCAACAACGCGGG GATCACGCGCGATGCTACATTTAAGAAAATGTCCTAT GAGCAATGGTCCCAAGTCATCGACACGAATTTAAAGA CTCTTTTTACCGTGACCCAGCCAGTATTTAATAAAATG CTTGAACAGAAGTCTGGCCGCATCGTAAACATTAGCT CTGTCAATGGTTTAAAAGGGCAATTTGGTCAAGCCAA CTACTCGGCCTCGAAAGCAGGGATTATCGGGTTTACT AAAGCATTGGCGCAGGAGGGTGCTCGCTCGAACATTT GCGTCAATGTCGTTGCTCCTGGTTACACAGCGACACCC ATGGTCACAGCAATGCGCGAGGATGTAATTAAGTCAA TCGAAGCTCAAATTCCCCTGCAACGTCTGGCAGCACC GGCGGAGATTGCGGCAGCGGTTATGTATTTGGTGAGT GAACACGGTGCATACGTGACGGGCGAAACTTTGAGTA TCAACGGCGGGCTGTACATGCACTAAAGGTGCTTTTA GTCTAGCGCTAGAGCAGGTACCATATTAATGAATCCA
AATTCCTTTCAGTTTAAAGAGAATATCTTACAGTTTTT CAGCGTGCACGACGATATTTGGAAAAAACTGCAGGAA TTTTACTATGGACAATCGCCCATCAATGAAGCGTTGGC GCAGTTAAATAAGGAAGACATGAGTTTATTCTTCGAG GCGTTATCAAAAAACCCTGCTCGTATGATGGAGATGC AGTGGTCCTGGTGGCAAGGGCAGATTCAAATTTACCA GAACGTGTTAATGCGTAGTGTAGCCAAGGACGTAGCC CCCTTTATCCAGCCAGAGTCCGGAGATCGTCGCTTCAA CTCGCCACTTTGGCAAGAACATCCAAATTTTGATTTAC TGAGTCAATCCTACTTGTTGTTTTCTCAGTTGGTTCAA AATATGGTGGATGTCGTTGAAGGAGTACCTGATAAGG TCCGCTATCGCATCCATTTCTTTACACGTCAGATGATC AATGCGTTGTCTCCTTCTAATTTCCTGTGGACGAACCC TGAAGTAATTCAACAGACGGTCGCTGAACAGGGTGAG AATTTAGTACGCGGGATGCAAGTATTTCACGATGATG TAATGAATTCGGGTAAATATTTGAGCATCCGTATGGT AAATAGCGACAGTTTCTCTCTTGGCAAGGACTTGGCG TATACGCCAGGAGCCGTAGTTTTCGAGAACGACATCT TTCAGCTTCTTCAATACGAAGCCACAACCGAGAACGT ATATCAAACCCCTATTCTTGTCGTACCTCCCTTCATCA ACAAGTACTACGTGCTGGACCTGCGCGAACAGAATAG CTTGGTTAATTGGCTGCGCCAACAAGGACATACGGTG TTTTTGATGTCGTGGCGTAACCCCAACGCAGAGCAGA AGGAGCTTACCTTCGCTGACTTAATTACCCAAGGATC GGTAGAAGCATTACGTGTTATCGAAGAAATCACGGGA GAGAAAGAAGCTAACTGTATTGGATATTGCATCGGTG GTACACTTCTGGCTGCTACCCAGGCATATTATGTAGCT AAACGCCTGAAAAATCACGTAAAGTCAGCGACTTATA TGGCGACGATTATTGATTTTGAGAACCCCGGCTCATTG GGTGTTTTCATTAATGAGCCGGTCGTAAGTGGACTTGA AAACCTTAATAATCAACTTGGTTACTTCGACGGGCGTC AACTTGCAGTGACATTTTCGTTGTTGCGCGAAAACACC TTGTATTGGAATTATTACATCGATAATTACTTGAAGGG TAAGGAACCGTCCGACTTTGACATCTTATACTGGAACT

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	CGGATGGTACGAATATCCCAGCAAAGATTCACAATTT CCTGTTACGTAACCTTTATCTTAACAACGAACTTATTT CTCCAAATGCCGTCAAAGTTAATGGTGTGGGTTTAAA CCTTTCGCGCGTGAAGACTCCATCATTCTTCATTGCTA CGCAGGAGGACCATATCGCATTGTGGGATACCTGTTT TCGCGGCGCGGATTACCTGGGGGGTGAGAGCACACTT GTGCTTGGGGAAAGCGGACACGTCGCCGGCATTGTCA ACCCGCCTTCTCGTAACAAGTATGGTTGTTACACGAAC GCCGCCAAGTTTGAAAATACCAAGCAATGGCTTGACG GTGCAGAATATCATCCCGAAAGCTGGTGGTTACGTTG GCAGGCATGGGTCACGCCTTATACTGGAGAGCAGGTT CCTGCGCGTAATTTGGGAAACGCACAGTACCCCAGTA TTGAAGCGGCCCCTGGGCGTTATGTGCTGGTAAACCT GTTTTAACGCTCACATACAAGCAATCTATAATTATTCA
CGGTATAAATGAAAGATGTTGTTATCGTAGCCGCTAA ACGCACTGCGATCGGTTCCTTTCTGGGGAGTCTGGCTT CCCTGAGCGCCCCTCAGTTGGGTCAGACGGCTATCCG CGCAGTTTTGGATTCTGCAAATGTGAAACCAGAACAA GTGGACCAAGTAATTATGGGGAATGTGCTGACCACCG GCGTTGGGCAAAATCCTGCTCGTCAGGCAGCAATCGC CGCTGGGATTCCTGTACAAGTTCCCGCCAGCACGCTTA ATGTAGTGTGTGGGTCCGGATTACGTGCCGTTCACCTG GCAGCTCAAGCCATCCAATGCGATGAAGCCGATATCG TCGTTGCCGGAGGTCAAGAATCAATGTCCCAGTCTGC TCATTACATGCAGCTTCGCAATGGCCAGAAAATGGGT AACGCACAGTTAGTCGATTCAATGGTGGCCGACGGCT TGACCGACGCGTATAATCAATACCAGATGGGTATCAC CGCGGAGAATATCGTCGAAAAACTTGGTCTTAATCGT GAAGAACAAGACCAGCTTGCTCTGACAAGTCAACAAC GTGCTGCAGCAGCGCAGGCTGCCGGAAAATTCAAGGA TGAAATTGCGGTCGTTTCGATTCCCCAGCGCAAAGGA GAGCCGGTCGTCTTCGCGGAAGACGAATATATCAAGG CCAATACCTCGTTGGAATCCTTGACGAAACTGCGTCC AGCATTCAAAAAAGACGGTTCTGTTACAGCCGGCAAC GCATCTGGCATTAATGATGGGGCAGCCGCGGTCCTGA TGATGTCCGCCGACAAAGCGGCTGAACTGGGCTTAAA GCCTTTAGCACGCATTAAAGGTTACGCGATGTCAGGA ATTGAGCCGGAAATCATGGGACTGGGTCCTGTAGACG CCGTTAAGAAAACCCTTAATAAGGCTGGTTGGTCCTT AGACCAGGTCGATCTGATCGAGGCCAATGAGGCTTTT GCTGCCCAAGCACTGGGAGTAGCCAAGGAGCTTGGGC TGGACCTGGACAAGGTAAATGTTAACGGAGGTGCGAT CGCGCTGGGACACCCGATCGGGGCTTCGGGTTGTCGT ATCTTGGTCACGTTATTACACGAAATGCAGCGTCGTG ATGCAAAGAAGGGTATCGCCACATTGTGTGTGGGAGG TGGAATGGGGGTGGCGCTTGCCGTTGAGCGCGATTAA
prpE sequence (comprised in	ATGTCTTTTAGCGAATTTTATCAGCGTTCGATTAACGA ACCGGAGAAGTTCTGGGCCGAGCAGGCCCGGCGTATT	SEQ ID

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GACTGGCAGACGCCCTTTACGCAAACGCTCGACCACA

GCAACCCGCCGTTTGCCCGTTGGTTTTGTGAAGGCCGA

ACCAACTTGTGTCACAACGCTATCGACCGCTGGCTGG

AGAAACAGCCAGAGGCGCTGGCATTGATTGCCGTCTC

TTCGGAAACAGAGGAAGAGCGTACCTTTACCTTCCGC

CAGTTACATGACGAAGTGAATGCGGTGGCGTCAATGC

TGCGCTCACTGGGCGTGCAGCGTGGCGATCGGGTGCT

GGTGTATATGCCGATGATTGCCGAAGCGCATATTACC

CTGCTGGCCTGCGCGCGCATTGGTGCTATTCACTCGGT

GGTGTTTGGGGGATTTGCTTCGCACAGCGTGGCAACG

CGAATTGATGACGCTAAACCGGTGCTGATTGTCTCGG

CTGATGCCGGGGCGCGCGGCGGTAAAATCATTCCGTA

TAAAAAATTGCTCGACGATGCGATAAGTCAGGCACAG

CATCAGCCGCGTCACGTTTTACTGGTGGATCGCGGGCT

GGCGAAAATGGCGCGCGTTAGCGGGCGGGATGTCGAT

TTCGCGTCGTTGCGCCATCAACACATCGGCGCGCGGG

TGCCGGTGGCATGGCTGGAATCCAACGAAACCTCCTG

CATTCTCTACACCTCCGGCACGACCGGCAAACCTAAA

GGTGTGCAGCGTGATGTCGGCGGATATGCGGTGGCGC

TGGCGACCTCGATGGACACCATTTTTGGCGGCAAAGC

GGGCGGCGTGTTCTTTTGTGCTTCGGATATCGGCTGGG

TGGTAGGGCATTCGTATATCGTTTACGCGCCGCTGCTG

GCGGGGATGGCGACTATCGTTTACGAAGGATTGCCGA

CCTGGCCGGACTGCGGCGTGTGGTGGAAAATTGTCGA

GAAATATCAGGTTAGCCGCATGTTCTCAGCGCCGACC

GCCATTCGCGTGCTGAAAAAATTCCCTACCGCTGAAA

TTCGCAAACACGATCTTTCGTCGCTGGAAGTGCTCTAT

CTGGCTGGAGAACCGCTGGACGAGCCGACCGCCAGTT

GGGTGAGCAATACGCTGGATGTGCCGGTCATCGACAA

CTACTGGCAGACCGAATCCGGCTGGCCGATTATGGCG

ATTGCTCGCGGTCTGGATGACAGACCGACGCGTCTGG

GAAGCCCCGGCGTGCCGATGTATGGCTATAACGTGCA

GTTGCTCAATGAAGTCACCGGCGAACCGTGTGGCGTC

AATGAGAAAGGGATGCTGGTAGTGGAGGGGCCATTGC

CGCCAGGCTGTATTCAAACCATCTGGGGCGACGACGA

CCGCTTTGTGAAGACGTACTGGTCGCTGTTTTCCCGTC

CGGTGTACGCCACTTTTGACTGGGGCATCCGCGATGCT

GACGGTTATCACTTTATTCTCGGGCGCACTGACGATGT

GATTAACGTTGCCGGACATCGGCTGGGTACGCGTGAG

ATTGAAGAGAGTATCTCCAGTCATCCGGGCGTTGCCG

AAGTGGCGGTGGTTGGGGTGAAAGATGCGCTGAAAG

GGCAGGTGGCGGTGGCGTTTGTCATTCCGAAAGAGAG

CGACAGTCTGGAAGACCGTGAGGTGGCGCACTCGCAA

GAGAAGGCGATTATGGCGCTGGTGGACAGCCAGATTG

GCAACTTTGGCCGCCCGGCGCACGTCTGGTTTGTCTCG

CAATTGCCAAAAACGCGATCCGGAAAAATGCTGCGCC

GCACGATCCAGGCGATTTGCGAAGGACGCGATCCTGG

GGATCTGACGACCATTGATGATCCGGCGTCGTTGGAT

CAGATCCGCCAGGCGATGGAAGAGTAG

NO:

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phaB sequence (comprised in the prpEPhaBCA construct shown in FIG. ID	ATGTCAGAGCAGAAAGTAGCTCTGGTTACCGGTGCGT TAGGTGGTATCGGAAGTGAGATCTGCCGCCAGCTTGT GACCGCCGGGTACAAGATTATCGCCACCGTTGTTCCA CGCGAAGAAGACCGCGAAAAACAATGGTTGCAAAGT GAGGGGTTTCAAGACTCTGATGTGCGTTTCGTATTAAC AGATTTAAACAATCACGAAGCTGCGACAGCGGCAATT CAAGAAGCGATTGCCGCCGAAGGACGCGTTGATGTAT TGGTCAACAACGCGGGGATCACGCGCGATGCTACATT TAAGAAAATGTCCTATGAGCAATGGTCCCAAGTCATC GACACGAATTTAAAGACTCTTTTTACCGTGACCCAGCC AGTATTTAATAAAATGCTTGAACAGAAGTCTGGCCGC ATCGTAAACATTAGCTCTGTCAATGGTTTAAAAGGGC AATTTGGTCAAGCCAACTACTCGGCCTCGAAAGCAGG GATTATCGGGTTTACTAAAGCATTGGCGCAGGAGGGT GCTCGCTCGAACATTTGCGTCAATGTCGTTGCTCCTGG TTACACAGCGACACCCATGGTCACAGCAATGCGCGAG GATGTAATTAAGTCAATCGAAGCTCAAATTCCCCTGC AACGTCTGGCAGCACCGGCGGAGATTGCGGCAGCGGT TATGTATTTGGTGAGTGAACACGGTGCATACGTGACG GGCGAAACTTTGAGTATCAACGGCGGGCTGTACATGC ACTAA	SEQ ID NO: 26
phaC sequence (comprised in the prpEPhaBCA construct shown in FIG. ID	ATGAATCCAAATTCCTTTCAGTTTAAAGAGAATATCTT ACAGTTTTTCAGCGTGCACGACGATATTTGGAAAAAA CTGCAGGAATTTTACTATGGACAATCGCCCATCAATG AAGCGTTGGCGCAGTTAAATAAGGAAGACATGAGTTT ATTCTTCGAGGCGTTATCAAAAAACCCTGCTCGTATGA TGGAGATGCAGTGGTCCTGGTGGCAAGGGCAGATTCA AATTTACCAGAACGTGTTAATGCGTAGTGTAGCCAAG GACGTAGCCCCCTTTATCCAGCCAGAGTCCGGAGATC GTCGCTTCAACTCGCCACTTTGGCAAGAACATCCAAA TTTTGATTTACTGAGTCAATCCTACTTGTTGTTTTCTCA GTTGGTTCAAAATATGGTGGATGTCGTTGAAGGAGTA CCTGATAAGGTCCGCTATCGCATCCATTTCTTTACACG TCAGATGATCAATGCGTTGTCTCCTTCTAATTTCCTGT GGACGAACCCTGAAGTAATTCAACAGACGGTCGCTGA ACAGGGTGAGAATTTAGTACGCGGGATGCAAGTATTT CACGATGATGTAATGAATTCGGGTAAATATTTGAGCA TCCGTATGGTAAATAGCGACAGTTTCTCTCTTGGCAAG GACTTGGCGTATACGCCAGGAGCCGTAGTTTTCGAGA ACGACATCTTTCAGCTTCTTCAATACGAAGCCACAACC GAGAACGTATATCAAACCCCTATTCTTGTCGTACCTCC CTTCATCAACAAGTACTACGTGCTGGACCTGCGCGAA CAGAATAGCTTGGTTAATTGGCTGCGCCAACAAGGAC ATACGGTGTTTTTGATGTCGTGGCGTAACCCCAACGCA GAGCAGAAGGAGCTTACCTTCGCTGACTTAATTACCC AAGGATCGGTAGAAGCATTACGTGTTATCGAAGAAAT CACGGGAGAGAAAGAAGCTAACTGTATTGGATATTGC ATCGGTGGTACACTTCTGGCTGCTACCCAGGCATATTA	SEQ ID NO: 27

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	TGTAGCTAAACGCCTGAAAAATCACGTAAAGTCAGCG ACTTATATGGCGACGATTATTGATTTTGAGAACCCCGG CTCATTGGGTGTTTTCATTAATGAGCCGGTCGTAAGTG GACTTGAAAACCTTAATAATCAACTTGGTTACTTCGAC GGGCGTCAACTTGCAGTGACATTTTCGTTGTTGCGCGA AAACACCTTGTATTGGAATTATTACATCGATAATTACT TGAAGGGTAAGGAACCGTCCGACTTTGACATCTTATA CTGGAACTCGGATGGTACGAATATCCCAGCAAAGATT CACAATTTCCTGTTACGTAACCTTTATCTTAACAACGA ACTTATTTCTCCAAATGCCGTCAAAGTTAATGGTGTGG GTTTAAACCTTTCGCGCGTGAAGACTCCATCATTCTTC ATTGCTACGCAGGAGGACCATATCGCATTGTGGGATA CCTGTTTTCGCGGCGCGGATTACCTGGGGGGTGAGAG CACACTTGTGCTTGGGGAAAGCGGACACGTCGCCGGC ATTGTCAACCCGCCTTCTCGTAACAAGTATGGTTGTTA CACGAACGCCGCCAAGTTTGAAAATACCAAGCAATGG CTTGACGGTGCAGAATATCATCCCGAAAGCTGGTGGT TACGTTGGCAGGCATGGGTCACGCCTTATACTGGAGA GCAGGTTCCTGCGCGTAATTTGGGAAACGCACAGTAC CCCAGTATTGAAGCGGCCCCTGGGCGTTATGTGCTGG TAAACCTGTTTTAA
phaA sequence (comprised in the prpEPhaBCA construct shown in FIG. ID	ATGAAAGATGTTGTTATCGTAGCCGCTAAACGCACTG CGATCGGTTCCTTTCTGGGGAGTCTGGCTTCCCTGAGC GCCCCTCAGTTGGGTCAGACGGCTATCCGCGCAGTTTT GGATTCTGCAAATGTGAAACCAGAACAAGTGGACCAA GTAATTATGGGGAATGTGCTGACCACCGGCGTTGGGC AAAATCCTGCTCGTCAGGCAGCAATCGCCGCTGGGAT TCCTGTACAAGTTCCCGCCAGCACGCTTAATGTAGTGT GTGGGTCCGGATTACGTGCCGTTCACCTGGCAGCTCA AGCCATCCAATGCGATGAAGCCGATATCGTCGTTGCC GGAGGTCAAGAATCAATGTCCCAGTCTGCTCATTACA TGCAGCTTCGCAATGGCCAGAAAATGGGTAACGCACA GTTAGTCGATTCAATGGTGGCCGACGGCTTGACCGAC GCGTATAATCAATACCAGATGGGTATCACCGCGGAGA ATATCGTCGAAAAACTTGGTCTTAATCGTGAAGAACA AGACCAGCTTGCTCTGACAAGTCAACAACGTGCTGCA GCAGCGCAGGCTGCCGGAAAATTCAAGGATGAAATTG CGGTCGTTTCGATTCCCCAGCGCAAAGGAGAGCCGGT CGTCTTCGCGGAAGACGAATATATCAAGGCCAATACC TCGTTGGAATCCTTGACGAAACTGCGTCCAGCATTCA AAAAAGACGGTTCTGTTACAGCCGGCAACGCATCTGG CATTAATGATGGGGCAGCCGCGGTCCTGATGATGTCC GCCGACAAAGCGGCTGAACTGGGCTTAAAGCCTTTAG CACGCATTAAAGGTTACGCGATGTCAGGAATTGAGCC GGAAATCATGGGACTGGGTCCTGTAGACGCCGTTAAG AAAACCCTTAATAAGGCTGGTTGGTCCTTAGACCAGG TCGATCTGATCGAGGCCAATGAGGCTTTTGCTGCCCA AGCACTGGGAGTAGCCAAGGAGCTTGGGCTGGACCTG	SEQ ID NO: 28

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GACAAGGTAAATGTTAACGGAGGTGCGATCGCGCTGG GACACCCGATCGGGGCTTCGGGTTGTCGTATCTTGGTC ACGTTATTACACGAAATGCAGCGTCGTGATGCAAAGA AGGGTATCGCCACATTGTGTGTGGGAGGTGGAATGGG GGTGGCGCTTGCCGTTGAGCGCGATTAA

[0519] The plasmid was transformed into E. coli DH5a as described herein to generate the plasmid pTet-prpE-PhaBCA.

[0520] In certain constructs, the prpE-PhaBCA operon is operably linked to a FNRresponsive promoter, which may be is further fused to a strong ribosome binding site sequence. For efficient translation, a 20-30 bp ribosome binding site was included for each synthetic gene in the operon. Each gene cassette and regulatory region construct is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.

[0521] In certain embodiments the construct is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., FIG. 32). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the propionate construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting E. coli Nissle bacteria are genetically engineered to express a propionate biosynthesis cassette and produce propionate.

Example 3. Construction of Plasmids Encoding Propionate Catabolism Enzymes (MMCA Pathway) [0522] The methylmalonyl-CoA pathway (MMCA) carries out reactions homologous to those in the mammalian pathway. Genes accA (from Streptomyces coelicolor), pccB (from Streptomyces coelicolor), mmcE (from Propionibacterium freudenreichii), and mutAB (from Propionibacterium freudenreichii) were codon-optimized for expression in E. coli Nissle. Two constructs were synthesized, the first with a cassette comprising prpE, pccB, accAl, under the control of an inducible Ptet promoter and the second with a cassette comprising

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PCT/US2016/044922 mmcE and mutAB under the control of a second inducible promoter, Para, (as shown in FIG

15C and FIG 16A and FIG. 16B).

[0523] The constructs were cloned into the plasmids pl5a-Kan (pTet-prpE- pccB,accAl) and an ColEl-Amp (pAra- mmcE-mutAB) by Golden Gate assembly, and transformed into E. coli DH5a as described herein. Sequences of MMCA pathway circuits are listed in Table 13.

Table 13. MMCA Pathway Circuit Sequences

Description	Sequence	SEQ ID NO
Construct comprising AraC (reverse orientation, lower case) and a mmcE-mutAmutB gene cassette under Para promoter (italics) (as shown in FIG. 15B and FIG. 16); ribosome binding sites are underlined ;. L3S2P11 terminator in italics; his terminator in bold; coding regions bold underlined	ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaa tactcgcgagaaatagagttgatcgtcaaaaccgacattgcgaccgacggt ggcgataggcatccgggtggtgctcaaaagcagcttcgcctgactgatgc gctggtcctcgcgccagcttaatacgctaatccctaactgctggcggaacaa atgcgacagacgcgacggcgacaggcagacatgctgtgcgacgctggc gatatcaaaattactgtctgccaggtgatcgctgatgtactgacaagcctcgc gtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcg ccgcagtaacaattgctcaagcagatttatcgccagcaattccgaatagcgc ccttccccttgtccggcattaatgatttgcccaaacaggtcgctgaaatgcgg ctggtgcgcttcatccgggcgaaagaaaccggtattggcaaatatcgacgg ccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccact ggtgataccattcgtgagcctccggatgacgaccgtagtgatgaatctctcc aggcgggaacagcaaaatatcacccggtcggcagacaaattctcgtccct gatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttc attcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcg gcgttaaacccgccaccagatgggcgttaaacgagtatcccggcagcagg ggatcattttgcgcttcagccatAC7777'CA7AC7'CCCGCC/17' TCAGAGAAGAAACCAATTGTCCATATTGCATCAG ACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCT CGCTAACCCAACCGGTAACCCCGCTTATTAAAAG CATTCTGTAACAAAGCGGGACCAAAGCCATGACA AAAACGCGTAACAAAAGTGTCTATAATCACGGCA GAAAAGTCCACATTGATTATTTGCACGGCGTCAC TAGCGGATCCAGCCTGACGCTTTTTTTCGCAACT CTCTACTGTTTCTCCATACCGGGAAACCACCGC GCCCAGCTTAATTTTATGAGTAACGAAGATT	SEQ ID NO: 29
TATTCATTTGCATCGACCACGTCGCGTATG
CGTGCCCGGATGCCGATGAAGCTTCTAAGT
ATTACCAGGAAACATTCGGTTGGCACGAGT
TGCACCGCGAAGAGAATCCAGAACAGGGC
GTGGTGGAAATTATGATGGCGCCTGCTGCG
AAATTGACGGAGCACATGACTCAGGTGCAA
GTTATGGCGCCTTTGAACGATGAGAGTACG
GTCGCGAAGTGGCTTGCGAAACACAATGG

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	GCGTGCTGGATTGCACCACATGGCATGGCG
TGTTGATGACATCGACGCAGTGTCCGCAAC
ACTTCGCGAGCGCGGTGTACAGTTGCTTTA
CGACGAGCCGAAACTGGGTACAGGTGGGA
ATCGTATCAACTTCATGCATCCGAAATCTG
GTAAAGGCGTGCTGATTGAACTGACCCAGT
ACCCCAAGAATTGATAAAGGTTTTTCCTAAG
ACGCTAGCGCATAAGGTCCACCAAATGTCAA
GTACAGACCAAGGCACGAACCCTGCTGACA
CGGATGATTTAACGCCAACCACATTATCCC
TGGCTGGTGATTTCCCTAAGGCTACGGAAG
AGCAGTGGGAGCGCGAGGTTGAAAAGGTG
TTGAACCGTGGGCGCCCACCCGAGAAGCA
GTTGACGTTTGCTGAATGTTTAAAACGTCT
TACTGTGCACACAGTAGATGGCATTGACAT
CGTTCCAATGTATCGCCCGAAGGATGCCCC
TAAGAAACTGGGGTATCCAGGGGTTGCTCC
CTTTACGCGTGGCACTACGGTTCGCAATGG
GGATATGGACGCTTGGGACGTTCGCGCCCT
GCACGAAGACCCTGATGAAAAATTCACGCG
CAAAGCTATTCTGGAGGGGCTGGAGCGCG
GCGTAACAAGTTTGCTTCTTCGTGTGGACC
CTGATGCAATCGCTCCCGAACACTTAGACG
AAGTGTTAAGTGACGTTTTGCTGGAAATGA
CCAAGGTTGAGGTGTTTTCCCGCTATGATC
AGGGAGCTGCGGCTGAAGCTCTTGTCTCGG
TATATGAGCGCAGCGACAAACCGGCTAAAG
ATTTGGCCTTAAATTTGGGACTGGACCCAA
TCGCATTTGCTGCACTTCAGGGCACTGAGC
CAGACTTGACCGTACTTGGTGATTGGGTTC
GTCGTTTGGCTAAATTCAGCCCAGACTCAC
GCGCTGTAACAATTGATGCTAATATTTATC
ACAACGCCGGTGCAGGCGACGTTGCCGAG
CTGGCCTGGGCACTTGCGACCGGAGCAGA
GTACGTCCGTGCGCTGGTAGAGCAAGGATT
CACCGCCACAGAGGCATTTGATACCATTAA
CTTCCGTGTGACAGCGACCCATGATCAATT
TTTAACGATTGCCCGCCTTCGTGCGTTACG
TGAAGCGTGGGCTCGTATCGGTGAGGTATT
CGGAGTAGATGAGGATAAACGTGGAGCGC
GCCAGAATGCTATTACGTCCTGGCGTGAAC
TGACACGCGAGGATCCCTATGTGAACATTT
TACGTGGAAGTATTGCCACGTTCTCTGCGT
CCGTTGGGGGCGCGGAGTCTATTACCACTT
TGCCATTCACGCAGGCATTGGGCCTTCCAG
AGGATGATTTTCCATTACGTATCGCACGTA
ATACAGGAATTGTCTTAGCTGAGGAGGTAA
ACATTGGGCGTGTAAATGACCCTGCCGGGG
GGTCATACTATGTGGAGAGCTTGACTCGTT

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	CTCTTGCAGATGCAGCATGGAAAGAGTTCC
AAGAGGTTGAAAAGTTGGGTGGTATGTCTA
AGGCCGTCATGACCGAACACGTCACGAAG
GTTTTAGATGCTTGCAACGCAGAGCGCGCG
AAGCGCTTGGCCAACCGCAAGCAACCTATT
ACGGCAGTTTCCGAATTTCCGATGATTGGC
GCACGCAGCATTGAGACGAAACCATTTCCG
GCTGCTCCGGCCCGTAAAGGGCTGGCATG
GCACCGCGATTCCGAAGTCTTCGAGCAACT
TATGGACCGCTCCACGTCAGTTTCAGAGCG
TCCGAAAGTATTTTTAGCATGTCTTGGGAC
GCGCCGCGATTTTGGAGGACGCGAAGGAT
TTTCATCTCCGGTTTGGCACATTGCCGGGA
TTGACACGCCTCAAGTAGAAGGTGGGACGA
CTGCTGAAATCGTGGAAGCGTTCAAAAAAT
CTGGGGCCCAAGTCGCCGATTTATGTTCGA
GTGCCAAAGTGTATGCTCAACAAGGCTTAG
AGGTGGCAAAGGCTCTGAAAGCGGCTGGG
GCTAAGGCGCTGTATTTGAGCGGAGCATTT
AAGGAGTTCGGAGACGATGCAGCGGAAGC
CGAAAAACTTATCGACGGACGCCTTTTCAT
GGGCATGGATGTCGTTGACACCCTGTCTTC
CACTTTAGATATCCTTGGAGTGGCGAAGTG
ATAAGCTTAAAACAATTTACATCCGGCCGGAA
CTTACTATGTCTACCTTACCTCGCTTTGACA
GTGTTGATTTAGGAAATGCGCCGGTCCCAG
CAGATGCTGCACGTCGTTTTGAGGAACTTG
CGGCGAAAGCCGGGACCGGCGAAGCCTGG
GAAACTGCGGAACAAATTCCAGTAGGCACG
TTGTTTAATGAAGACGTATACAAGGACATG
GATTGGCTTGATACTTACGCTGGCATTCCT
CCCTTCGTCCATGGTCCGTACGCTACTATG
TATGCATTTCGTCCTTGGACCATTCGCCAA
TATGCCGGTTTTTCGACTGCAAAGGAGTCA
AACGCATTTTACCGTCGTAATTTGGCTGCA
GGCCAGAAAGGTCTTAGTGTTGCTTTTGAC
TTACCCACTCACCGCGGTTATGATTCCGAC
AACCCCCGCGTGGCCGGAGATGTTGGTATG
GCCGGTGTGGCTATCGATTCGATTTATGAC
ATGCGTGAGCTGTTCGCCGGCATCCCATTA
GATCAGATGAGCGTGTCGATGACAATGAAC
GGTGCTGTCTTGCCGATTTTGGCTCTTTAT
GTGGTTACGGCGGAGGAGCAAGGCGTGAA
GCCAGAACAACTGGCGGGTACTATTCAAAA
TGATATTCTGAAGGAATTTATGGTTCGTAA
TACATATATTTACCCGCCGCAACCTAGTAT
GCGCATTATCAGCGAGATTTTTGCATACAC
ATCAGCAAACATGCCGAAGTGGAACTCCAT
TAGTATCAGCGGCTATCATATGCAGGAGGC

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	TGGAGCGACTGCGGATATCGAGATGGCGT
ATACCTTAGCTGATGGAGTTGATTACATCC
GTGCTGGTGAGTCAGTAGGACTTAATGTGG
ACCAATTTGCTCCACGCCTGTCCTTCTTCT
GGGGCATTGGTATGAACTTTTTCATGGAGG
TAGCGAAGTTACGCGCTGCCCGTATGCTGT
GGGCGAAGCTTGTCCACCAGTTCGGCCCGA
AAAACCCGAAGAGTATGTCTCTGCGCACGC
ACTCTCAAACATCGGGTTGGTCTTTGACAG
CTCAAGACGTATATAATAACGTTGTACGTA
CATGCATCGAAGCCATGGCTGCTACTCAAG
GCCATACTCAATCACTTCATACAAATTCGTT
GGATGAAGCCATTGCATTGCCTACGGACTT
TTCAGCCCGCATTGCCCGCAATACTCAATT
ATTTCTGCAACAAGAGAGCGGGACGACTCG
TGTGATCGACCCTTGGTCAGGTTCCGCATA
CGTCGAAGAGTTGACTTGGGATTTAGCTCG
TAAAGCCTGGGGGCATATTCAGGAGGTTGA
GAAGGTGGGGGGCATGGCTAAGGCAATCG
AGAAGGGGATTCCGAAGATGCGCATTGAG
GAGGCAGCCGCCCGTACCCAAGCACGTATT
GATTCGGGACGCCAGCCATTAATTGGGGTC
AATAAATACCGTCTGGAGCACGAACCACCC
CTGGATGTGTTGAAGGTAGACAATAGCACC
GTGTTAGCTGAGCAAAAGGCCAAACTTGTT
AAATTGCGCGCAGAACGCGACCCAGAAAA
GGTCAAGGCTGCTCTGGACAAAATCACTTG
GGCGGCTGGCAATCCTGATGATAAAGACCC
TGATCGCAACTTATTAAAGCTGTGCATTGA
TGCGGGGCGCGCGATGGCAACGGTAGGAG
AGATGAGTGACGCTTTAGAGAAAGTTTTTG
GGCGCTACACAGCGCAAATTCGCACTATTT
CAGGAGTATATTCAAAAGAAGTCAAAAACA
CTCCGGAAGTCGAGGAGGCTCGCGAACTG
GTAGAAGAGTTTGAGCAGGCCGAAGGCCG
TCGCCCACGTATCCTGCTGGCTAAAATGGG
GCAGGACGGTCATGACCGTGGGCAAAAGG
TCATCGCGACTGCATACGCCGATTTGGGAT
TTGACGTGGACGTTGGCCCGTTATTCCAAA
CTCCCGAGGAAACTGCTCGCCAAGCCGTCG
AAGCCGATGTGCACGTAGTGGGGGTGAGC
TCTCTGGCGGGAGGGCATCTTACGCTTGTG
CCTGCGCTTCGCAAAGAGCTGGACAAGTTG
GGTCGTCCAGATATTCTGATTACCGTAGGA
GGGGTTATTCCCGAGCAGGACTTCGATGAG
CTTCGTAAGGATGGCGCTGTTGAAATCTAC
ACACCGGGGACGGTCATTCCAGAATCGGCT
ATCTCTTTAGTTAAAAAATTGCGCGCCTCC
CTGGATGCTTGATAAGGAGCrCGG7ACCAAAT

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	TCCAGAAAAGAGACGCTTTCGAGCGTCTTTTTTC GTTTTGGrCCGCGCAATAAAAAAGCCCCCGG AAGGTGATCTTCCGGGGGCTTTCTCATGCG TT
Construct comprising a mmcE-mutA-mutB gene cassette under the control of the Para promoter (as shown in FIG. 15B and FIG. 16) ribosome binding sites are underlined coding regions bold underlined	ACTTTTCATACTCCCGCCATTCAGAGAAGAAACC AATTGTCCATATTGCATCAGACATTGCCGTCACTG CGTCTTTTACTGGCTCTTCTCGCTAACCCAACCG GTAACCCCGCTTATTAAAAGCATTCTGTAACAAAG CGGGACCAAAGCCATGACAAAAACGCGTAACAAA AGTGTCTATAATCACGGCAGAAAAGTCCACATTG ATTATTTGCACGGCGTCACACTTTGCTATGCCATA GCATTTTTATCCATAAGATTAGCGGATCCAGCCT GACGCTTTTTTTCGCAACTCTCTACTGTTTCTCCA 7ACCGGGAAACCACCGCGCCCAGCTTAATTTT ATGAGTAACGAAGATTTATTCATTTGCATC	SEQ ID NO: 30
GACCACGTCGCGTATGCGTGCCCGGATGCC
GATGAAGCTTCTAAGTATTACCAGGAAACA
TTCGGTTGGCACGAGTTGCACCGCGAAGAG
AATCCAGAACAGGGCGTGGTGGAAATTATG
ATGGCGCCTGCTGCGAAATTGACGGAGCAC
ATGACTCAGGTGCAAGTTATGGCGCCTTTG
AACGATGAGAGTACGGTCGCGAAGTGGCTT
GCGAAACACAATGGGCGTGCTGGATTGCAC
CACATGGCATGGCGTGTTGATGACATCGAC
GCAGTGTCCGCAACACTTCGCGAGCGCGGT
GTACAGTTGCTTTACGACGAGCCGAAACTG
GGTACAGGTGGGAATCGTATCAACTTCATG
CATCCGAAATCTGGTAAAGGCGTGCTGATT
GAACTGACCCAGTACCCCAAGAATTGATAA
AGGTTTTTCCTAAGACGCTAGCGCATAAGGTC CACCAAATGTCAAGTACAGACCAAGGCACG
AACCCTGCTGACACGGATGATTTAACGCCA
ACCACATTATCCCTGGCTGGTGATTTCCCT
AAGGCTACGGAAGAGCAGTGGGAGCGCGA
GGTTGAAAAGGTGTTGAACCGTGGGCGCC
CACCCGAGAAGCAGTTGACGTTTGCTGAAT
GTTTAAAACGTCTTACTGTGCACACAGTAG
ATGGCATTGACATCGTTCCAATGTATCGCC
CGAAGGATGCCCCTAAGAAACTGGGGTATC
CAGGGGTTGCTCCCTTTACGCGTGGCACTA
CGGTTCGCAATGGGGATATGGACGCTTGG
GACGTTCGCGCCCTGCACGAAGACCCTGAT
GAAAAATTCACGCGCAAAGCTATTCTGGAG
GGGCTGGAGCGCGGCGTAACAAGTTTGCTT
CTTCGTGTGGACCCTGATGCAATCGCTCCC
GAACACTTAGACGAAGTGTTAAGTGACGTT
TTGCTGGAAATGACCAAGGTTGAGGTGTTT
TCCCGCTATGATCAGGGAGCTGCGGCTGAA

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	GCTCTTGTCTCGGTATATGAGCGCAGCGAC
AAACCGGCTAAAGATTTGGCCTTAAATTTG
GGACTGGACCCAATCGCATTTGCTGCACTT
CAGGGCACTGAGCCAGACTTGACCGTACTT
GGTGATTGGGTTCGTCGTTTGGCTAAATTC
AGCCCAGACTCACGCGCTGTAACAATTGAT
GCTAATATTTATCACAACGCCGGTGCAGGC
GACGTTGCCGAGCTGGCCTGGGCACTTGC
GACCGGAGCAGAGTACGTCCGTGCGCTGG
TAGAGCAAGGATTCACCGCCACAGAGGCAT
TTGATACCATTAACTTCCGTGTGACAGCGA
CCCATGATCAATTTTTAACGATTGCCCGCC
TTCGTGCGTTACGTGAAGCGTGGGCTCGTA
TCGGTGAGGTATTCGGAGTAGATGAGGATA
AACGTGGAGCGCGCCAGAATGCTATTACGT
CCTGGCGTGAACTGACACGCGAGGATCCCT
ATGTGAACATTTTACGTGGAAGTATTGCCA
CGTTCTCTGCGTCCGTTGGGGGCGCGGAGT
CTATTACCACTTTGCCATTCACGCAGGCAT
TGGGCCTTCCAGAGGATGATTTTCCATTAC
GTATCGCACGTAATACAGGAATTGTCTTAG
CTGAGGAGGTAAACATTGGGCGTGTAAATG
ACCCTGCCGGGGGGTCATACTATGTGGAGA
GCTTGACTCGTTCTCTTGCAGATGCAGCAT
GGAAAGAGTTCCAAGAGGTTGAAAAGTTGG
GTGGTATGTCTAAGGCCGTCATGACCGAAC
ACGTCACGAAGGTTTTAGATGCTTGCAACG
CAGAGCGCGCGAAGCGCTTGGCCAACCGC
AAGCAACCTATTACGGCAGTTTCCGAATTT
CCGATGATTGGCGCACGCAGCATTGAGACG
AAACCATTTCCGGCTGCTCCGGCCCGTAAA
GGGCTGGCATGGCACCGCGATTCCGAAGT
CTTCGAGCAACTTATGGACCGCTCCACGTC
AGTTTCAGAGCGTCCGAAAGTATTTTTAGC
ATGTCTTGGGACGCGCCGCGATTTTGGAGG
ACGCGAAGGATTTTCATCTCCGGTTTGGCA
CATTGCCGGGATTGACACGCCTCAAGTAGA
AGGTGGGACGACTGCTGAAATCGTGGAAG
CGTTCAAAAAATCTGGGGCCCAAGTCGCCG
ATTTATGTTCGAGTGCCAAAGTGTATGCTC
AACAAGGCTTAGAGGTGGCAAAGGCTCTGA
AAGCGGCTGGGGCTAAGGCGCTGTATTTGA
GCGGAGCATTTAAGGAGTTCGGAGACGAT
GCAGCGGAAGCCGAAAAACTTATCGACGG
ACGCCTTTTCATGGGCATGGATGTCGTTGA
CACCCTGTCTTCCACTTTAGATATCCTTGG
AGTGGCGAAGTGATAAGCTTAAAACAATTTA
CATCCGGCCGGAACTTACTATGTCTACCTTA
CCTCGCTTTGACAGTGTTGATTTAGGAAAT

-203WO 2017/023818

PCT/US2016/044922

	GCGCCGGTCCCAGCAGATGCTGCACGTCGT
TTTGAGGAACTTGCGGCGAAAGCCGGGAC
CGGCGAAGCCTGGGAAACTGCGGAACAAA
TTCCAGTAGGCACGTTGTTTAATGAAGACG
TATACAAGGACATGGATTGGCTTGATACTT
ACGCTGGCATTCCTCCCTTCGTCCATGGTC
CGTACGCTACTATGTATGCATTTCGTCCTT
GGACCATTCGCCAATATGCCGGTTTTTCGA
CTGCAAAGGAGTCAAACGCATTTTACCGTC
GTAATTTGGCTGCAGGCCAGAAAGGTCTTA
GTGTTGCTTTTGACTTACCCACTCACCGCG
GTTATGATTCCGACAACCCCCGCGTGGCCG
GAGATGTTGGTATGGCCGGTGTGGCTATCG
ATTCGATTTATGACATGCGTGAGCTGTTCG
CCGGCATCCCATTAGATCAGATGAGCGTGT
CGATGACAATGAACGGTGCTGTCTTGCCGA
TTTTGGCTCTTTATGTGGTTACGGCGGAGG
AGCAAGGCGTGAAGCCAGAACAACTGGCG
GGTACTATTCAAAATGATATTCTGAAGGAA
TTTATGGTTCGTAATACATATATTTACCCGC
CGCAACCTAGTATGCGCATTATCAGCGAGA
TTTTTGCATACACATCAGCAAACATGCCGA
AGTGGAACTCCATTAGTATCAGCGGCTATC
ATATGCAGGAGGCTGGAGCGACTGCGGAT
ATCGAGATGGCGTATACCTTAGCTGATGGA
GTTGATTACATCCGTGCTGGTGAGTCAGTA
GGACTTAATGTGGACCAATTTGCTCCACGC
CTGTCCTTCTTCTGGGGCATTGGTATGAAC
TTTTTCATGGAGGTAGCGAAGTTACGCGCT
GCCCGTATGCTGTGGGCGAAGCTTGTCCAC
CAGTTCGGCCCGAAAAACCCGAAGAGTATG
TCTCTGCGCACGCACTCTCAAACATCGGGT
TGGTCTTTGACAGCTCAAGACGTATATAAT
AACGTTGTACGTACATGCATCGAAGCCATG
GCTGCTACTCAAGGCCATACTCAATCACTT
CATACAAATTCGTTGGATGAAGCCATTGCA
TTGCCTACGGACTTTTCAGCCCGCATTGCC
CGCAATACTCAATTATTTCTGCAACAAGAG
AGCGGGACGACTCGTGTGATCGACCCTTGG
TCAGGTTCCGCATACGTCGAAGAGTTGACT
TGGGATTTAGCTCGTAAAGCCTGGGGGCAT
ATTCAGGAGGTTGAGAAGGTGGGGGGCAT
GGCTAAGGCAATCGAGAAGGGGATTCCGA
AGATGCGCATTGAGGAGGCAGCCGCCCGT
ACCCAAGCACGTATTGATTCGGGACGCCAG
CCATTAATTGGGGTCAATAAATACCGTCTG
GAGCACGAACCACCCCTGGATGTGTTGAAG
GTAGACAATAGCACCGTGTTAGCTGAGCAA
AAGGCCAAACTTGTTAAATTGCGCGCAGAA

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	CGCGACCCAGAAAAGGTCAAGGCTGCTCTG
GACAAAATCACTTGGGCGGCTGGCAATCCT
GATGATAAAGACCCTGATCGCAACTTATTA
AAGCTGTGCATTGATGCGGGGCGCGCGAT
GGCAACGGTAGGAGAGATGAGTGACGCTT
TAGAGAAAGTTTTTGGGCGCTACACAGCGC
AAATTCGCACTATTTCAGGAGTATATTCAA
AAGAAGTCAAAAACACTCCGGAAGTCGAGG
AGGCTCGCGAACTGGTAGAAGAGTTTGAGC
AGGCCGAAGGCCGTCGCCCACGTATCCTGC
TGGCTAAAATGGGGCAGGACGGTCATGAC
CGTGGGCAAAAGGTCATCGCGACTGCATAC
GCCGATTTGGGATTTGACGTGGACGTTGGC
CCGTTATTCCAAACTCCCGAGGAAACTGCT
CGCCAAGCCGTCGAAGCCGATGTGCACGTA
GTGGGGGTGAGCTCTCTGGCGGGAGGGCA
TCTTACGCTTGTGCCTGCGCTTCGCAAAGA
GCTGGACAAGTTGGGTCGTCCAGATATTCT
GATTACCGTAGGAGGGGTTATTCCCGAGCA
GGACTTCGATGAGCTTCGTAAGGATGGCGC
TGTTGAAATCTACACACCGGGGACGGTCAT
TCCAGAATCGGCTATCTCTTTAGTTAAAAA
ATTGCGCGCCTCCCTGGATGCT
Construct comprising a mmcE-mutA-mutB gene cassette; (as shown in FIG. 15B and FIG. 16) ribosome binding sites are underlined	GGGAAACCACCGCGCCCAGCTTAATTTTATGA GTAACGAAGATTTATTCATTTGCATCGACC	SEQ ID NO: 31
ACGTCGCGTATGCGTGCCCGGATGCCGATG AAGCTTCTAAGTATTACCAGGAAACATTCG
GTTGGCACGAGTTGCACCGCGAAGAGAATC
CAGAACAGGGCGTGGTGGAAATTATGATG
GCGCCTGCTGCGAAATTGACGGAGCACATG
ACTCAGGTGCAAGTTATGGCGCCTTTGAAC
GATGAGAGTACGGTCGCGAAGTGGCTTGC
GAAACACAATGGGCGTGCTGGATTGCACCA
CATGGCATGGCGTGTTGATGACATCGACGC
AGTGTCCGCAACACTTCGCGAGCGCGGTGT
ACAGTTGCTTTACGACGAGCCGAAACTGGG
TACAGGTGGGAATCGTATCAACTTCATGCA
TCCGAAATCTGGTAAAGGCGTGCTGATTGA
ACTGACCCAGTACCCCAAGAATTGATAAAG _{GTTTTTCCTAAGACGCTAGCGCATAAGGTCCA} CCAAATGTCAAGTACAGACCAAGGCACGAA
CCCTGCTGACACGGATGATTTAACGCCAAC
CACATTATCCCTGGCTGGTGATTTCCCTAA
GGCTACGGAAGAGCAGTGGGAGCGCGAGG
TTGAAAAGGTGTTGAACCGTGGGCGCCCAC
CCGAGAAGCAGTTGACGTTTGCTGAATGTT
TAAAACGTCTTACTGTGCACACAGTAGATG
GCATTGACATCGTTCCAATGTATCGCCCGA

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	AGGATGCCCCTAAGAAACTGGGGTATCCAG
GGGTTGCTCCCTTTACGCGTGGCACTACGG
TTCGCAATGGGGATATGGACGCTTGGGACG
TTCGCGCCCTGCACGAAGACCCTGATGAAA
AATTCACGCGCAAAGCTATTCTGGAGGGGC
TGGAGCGCGGCGTAACAAGTTTGCTTCTTC
GTGTGGACCCTGATGCAATCGCTCCCGAAC
ACTTAGACGAAGTGTTAAGTGACGTTTTGC
TGGAAATGACCAAGGTTGAGGTGTTTTCCC
GCTATGATCAGGGAGCTGCGGCTGAAGCTC
TTGTCTCGGTATATGAGCGCAGCGACAAAC
CGGCTAAAGATTTGGCCTTAAATTTGGGAC
TGGACCCAATCGCATTTGCTGCACTTCAGG
GCACTGAGCCAGACTTGACCGTACTTGGTG
ATTGGGTTCGTCGTTTGGCTAAATTCAGCC
CAGACTCACGCGCTGTAACAATTGATGCTA
ATATTTATCACAACGCCGGTGCAGGCGACG
TTGCCGAGCTGGCCTGGGCACTTGCGACCG
GAGCAGAGTACGTCCGTGCGCTGGTAGAG
CAAGGATTCACCGCCACAGAGGCATTTGAT
ACCATTAACTTCCGTGTGACAGCGACCCAT
GATCAATTTTTAACGATTGCCCGCCTTCGT
GCGTTACGTGAAGCGTGGGCTCGTATCGGT
GAGGTATTCGGAGTAGATGAGGATAAACGT
GGAGCGCGCCAGAATGCTATTACGTCCTGG
CGTGAACTGACACGCGAGGATCCCTATGTG
AACATTTTACGTGGAAGTATTGCCACGTTC
TCTGCGTCCGTTGGGGGCGCGGAGTCTATT
ACCACTTTGCCATTCACGCAGGCATTGGGC
CTTCCAGAGGATGATTTTCCATTACGTATC
GCACGTAATACAGGAATTGTCTTAGCTGAG
GAGGTAAACATTGGGCGTGTAAATGACCCT
GCCGGGGGGTCATACTATGTGGAGAGCTT
GACTCGTTCTCTTGCAGATGCAGCATGGAA
AGAGTTCCAAGAGGTTGAAAAGTTGGGTGG
TATGTCTAAGGCCGTCATGACCGAACACGT
CACGAAGGTTTTAGATGCTTGCAACGCAGA
GCGCGCGAAGCGCTTGGCCAACCGCAAGC
AACCTATTACGGCAGTTTCCGAATTTCCGA
TGATTGGCGCACGCAGCATTGAGACGAAAC
CATTTCCGGCTGCTCCGGCCCGTAAAGGGC
TGGCATGGCACCGCGATTCCGAAGTCTTCG
AGCAACTTATGGACCGCTCCACGTCAGTTT
CAGAGCGTCCGAAAGTATTTTTAGCATGTC
TTGGGACGCGCCGCGATTTTGGAGGACGC
GAAGGATTTTCATCTCCGGTTTGGCACATT
GCCGGGATTGACACGCCTCAAGTAGAAGGT
GGGACGACTGCTGAAATCGTGGAAGCGTTC
AAAAAATCTGGGGCCCAAGTCGCCGATTTA

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	TGTTCGAGTGCCAAAGTGTATGCTCAACAA
GGCTTAGAGGTGGCAAAGGCTCTGAAAGC
GGCTGGGGCTAAGGCGCTGTATTTGAGCG
GAGCATTTAAGGAGTTCGGAGACGATGCAG
CGGAAGCCGAAAAACTTATCGACGGACGCC
TTTTCATGGGCATGGATGTCGTTGACACCC
TGTCTTCCACTTTAGATATCCTTGGAGTGG
CGAAGTGATAAGCTTAAAACAATTTACATCC
GGCCGGAACTTACTATGTCTACCTTACCTCG
CTTTGACAGTGTTGATTTAGGAAATGCGCC
GGTCCCAGCAGATGCTGCACGTCGTTTTGA
GGAACTTGCGGCGAAAGCCGGGACCGGCG
AAGCCTGGGAAACTGCGGAACAAATTCCAG
TAGGCACGTTGTTTAATGAAGACGTATACA
AGGACATGGATTGGCTTGATACTTACGCTG
GCATTCCTCCCTTCGTCCATGGTCCGTACG
CTACTATGTATGCATTTCGTCCTTGGACCA
TTCGCCAATATGCCGGTTTTTCGACTGCAA
AGGAGTCAAACGCATTTTACCGTCGTAATT
TGGCTGCAGGCCAGAAAGGTCTTAGTGTTG
CTTTTGACTTACCCACTCACCGCGGTTATG
ATTCCGACAACCCCCGCGTGGCCGGAGATG
TTGGTATGGCCGGTGTGGCTATCGATTCGA
TTTATGACATGCGTGAGCTGTTCGCCGGCA
TCCCATTAGATCAGATGAGCGTGTCGATGA
CAATGAACGGTGCTGTCTTGCCGATTTTGG
CTCTTTATGTGGTTACGGCGGAGGAGCAAG
GCGTGAAGCCAGAACAACTGGCGGGTACT
ATTCAAAATGATATTCTGAAGGAATTTATG
GTTCGTAATACATATATTTACCCGCCGCAA
CCTAGTATGCGCATTATCAGCGAGATTTTT
GCATACACATCAGCAAACATGCCGAAGTGG
AACTCCATTAGTATCAGCGGCTATCATATG
CAGGAGGCTGGAGCGACTGCGGATATCGA
GATGGCGTATACCTTAGCTGATGGAGTTGA
TTACATCCGTGCTGGTGAGTCAGTAGGACT
TAATGTGGACCAATTTGCTCCACGCCTGTC
CTTCTTCTGGGGCATTGGTATGAACTTTTT
CATGGAGGTAGCGAAGTTACGCGCTGCCC
GTATGCTGTGGGCGAAGCTTGTCCACCAGT
TCGGCCCGAAAAACCCGAAGAGTATGTCTC
TGCGCACGCACTCTCAAACATCGGGTTGGT
CTTTGACAGCTCAAGACGTATATAATAACG
TTGTACGTACATGCATCGAAGCCATGGCTG
CTACTCAAGGCCATACTCAATCACTTCATA
CAAATTCGTTGGATGAAGCCATTGCATTGC
CTACGGACTTTTCAGCCCGCATTGCCCGCA
ATACTCAATTATTTCTGCAACAAGAGAGCG
GGACGACTCGTGTGATCGACCCTTGGTCAG

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	GTTCCGCATACGTCGAAGAGTTGACTTGGG
ATTTAGCTCGTAAAGCCTGGGGGCATATTC
AGGAGGTTGAGAAGGTGGGGGGCATGGCT
AAGGCAATCGAGAAGGGGATTCCGAAGAT
GCGCATTGAGGAGGCAGCCGCCCGTACCC
AAGCACGTATTGATTCGGGACGCCAGCCAT
TAATTGGGGTCAATAAATACCGTCTGGAGC
ACGAACCACCCCTGGATGTGTTGAAGGTAG
ACAATAGCACCGTGTTAGCTGAGCAAAAGG
CCAAACTTGTTAAATTGCGCGCAGAACGCG
ACCCAGAAAAGGTCAAGGCTGCTCTGGACA
AAATCACTTGGGCGGCTGGCAATCCTGATG
ATAAAGACCCTGATCGCAACTTATTAAAGC
TGTGCATTGATGCGGGGCGCGCGATGGCA
ACGGTAGGAGAGATGAGTGACGCTTTAGA
GAAAGTTTTTGGGCGCTACACAGCGCAAAT
TCGCACTATTTCAGGAGTATATTCAAAAGA
AGTCAAAAACACTCCGGAAGTCGAGGAGG
CTCGCGAACTGGTAGAAGAGTTTGAGCAGG
CCGAAGGCCGTCGCCCACGTATCCTGCTGG
CTAAAATGGGGCAGGACGGTCATGACCGT
GGGCAAAAGGTCATCGCGACTGCATACGCC
GATTTGGGATTTGACGTGGACGTTGGCCCG
TTATTCCAAACTCCCGAGGAAACTGCTCGC
CAAGCCGTCGAAGCCGATGTGCACGTAGTG
GGGGTGAGCTCTCTGGCGGGAGGGCATCT
TACGCTTGTGCCTGCGCTTCGCAAAGAGCT
GGACAAGTTGGGTCGTCCAGATATTCTGAT
TACCGTAGGAGGGGTTATTCCCGAGCAGGA
CTTCGATGAGCTTCGTAAGGATGGCGCTGT
TGAAATCTACACACCGGGGACGGTCATTCC
AGAATCGGCTATCTCTTTAGTTAAAAAATT
GCGCGCCTCCCTGGATGCT
mmcE sequence (comprised in the mmcEmutA-mutB construct shown in FIG. 15B and FIG. 16)	ATGAGTAACGAAGATTTATTCATTTGCATCGA CCACGTCGCGTATGCGTGCCCGGATGCCGATG AAGCTTCTAAGTATTACCAGGAAACATTCGGT TGGCACGAGTTGCACCGCGAAGAGAATCCAG AACAGGGCGTGGTGGAAATTATGATGGCGCC TGCTGCGAAATTGACGGAGCACATGACTCAG GTGCAAGTTATGGCGCCTTTGAACGATGAGAG TACGGTCGCGAAGTGGCTTGCGAAACACAAT GGGCGTGCTGGATTGCACCACATGGCATGGC GTGTTGATGACATCGACGCAGTGTCCGCAACA CTTCGCGAGCGCGGTGTACAGTTGCTTTACGA CGAGCCGAAACTGGGTACAGGTGGGAATCGT ATCAACTTCATGCATCCGAAATCTGGTAAAGG CGTGCTGATTGAACTGACCCAGTACCCCAAGA	SEQ ID NO: 32

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	ATTGA
mutA sequence (comprised in the mmcE-mutA-mutB construct shown in FIG. 15B and FIG. 16)	ATGTCAAGTACAGACCAAGGCACGAACCCTG CTGACACGGATGATTTAACGCCAACCACATTA TCCCTGGCTGGTGATTTCCCTAAGGCTACGGA AGAGCAGTGGGAGCGCGAGGTTGAAAAGGTG TTGAACCGTGGGCGCCCACCCGAGAAGCAGT TGACGTTTGCTGAATGTTTAAAACGTCTTACT GTGCACACAGTAGATGGCATTGACATCGTTCC AATGTATCGCCCGAAGGATGCCCCTAAGAAA CTGGGGTATCCAGGGGTTGCTCCCTTTACGCG TGGCACTACGGTTCGCAATGGGGATATGGAC GCTTGGGACGTTCGCGCCCTGCACGAAGACCC TGATGAAAAATTCACGCGCAAAGCTATTCTGG AGGGGCTGGAGCGCGGCGTAACAAGTTTGCT TCTTCGTGTGGACCCTGATGCAATCGCTCCCG AACACTTAGACGAAGTGTTAAGTGACGTTTTG CTGGAAATGACCAAGGTTGAGGTGTTTTCCCG CTATGATCAGGGAGCTGCGGCTGAAGCTCTTG TCTCGGTATATGAGCGCAGCGACAAACCGGCT AAAGATTTGGCCTTAAATTTGGGACTGGACCC AATCGCATTTGCTGCACTTCAGGGCACTGAGC CAGACTTGACCGTACTTGGTGATTGGGTTCGT CGTTTGGCTAAATTCAGCCCAGACTCACGCGC TGTAACAATTGATGCTAATATTTATCACAACG CCGGTGCAGGCGACGTTGCCGAGCTGGCCTG GGCACTTGCGACCGGAGCAGAGTACGTCCGT GCGCTGGTAGAGCAAGGATTCACCGCCACAG AGGCATTTGATACCATTAACTTCCGTGTGACA GCGACCCATGATCAATTTTTAACGATTGCCCG CCTTCGTGCGTTACGTGAAGCGTGGGCTCGTA TCGGTGAGGTATTCGGAGTAGATGAGGATAA ACGTGGAGCGCGCCAGAATGCTATTACGTCCT GGCGTGAACTGACACGCGAGGATCCCTATGT GAACATTTTACGTGGAAGTATTGCCACGTTCT CTGCGTCCGTTGGGGGCGCGGAGTCTATTACC ACTTTGCCATTCACGCAGGCATTGGGCCTTCC AGAGGATGATTTTCCATTACGTATCGCACGTA ATACAGGAATTGTCTTAGCTGAGGAGGTAAA CATTGGGCGTGTAAATGACCCTGCCGGGGGGT CATACTATGTGGAGAGCTTGACTCGTTCTCTT GCAGATGCAGCATGGAAAGAGTTCCAAGAGG TTGAAAAGTTGGGTGGTATGTCTAAGGCCGTC ATGACCGAACACGTCACGAAGGTTTTAGATGC TTGCAACGCAGAGCGCGCGAAGCGCTTGGCC AACCGCAAGCAACCTATTACGGCAGTTTCCGA ATTTCCGATGATTGGCGCACGCAGCATTGAGA CGAAACCATTTCCGGCTGCTCCGGCCCGTAAA GGGCTGGCATGGCACCGCGATTCCGAAGTCTT	SEQ ID NO: 33

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PCT/US2016/044922

	CGAGCAACTTATGGACCGCTCCACGTCAGTTT CAGAGCGTCCGAAAGTATTTTTAGCATGTCTT GGGACGCGCCGCGATTTTGGAGGACGCGAAG GATTTTCATCTCCGGTTTGGCACATTGCCGGG ATTGACACGCCTCAAGTAGAAGGTGGGACGA CTGCTGAAATCGTGGAAGCGTTCAAAAAATCT GGGGCCCAAGTCGCCGATTTATGTTCGAGTGC CAAAGTGTATGCTCAACAAGGCTTAGAGGTG GCAAAGGCTCTGAAAGCGGCTGGGGCTAAGG CGCTGTATTTGAGCGGAGCATTTAAGGAGTTC GGAGACGATGCAGCGGAAGCCGAAAAACTTA TCGACGGACGCCTTTTCATGGGCATGGATGTC GTTGACACCCTGTCTTCCACTTTAGATATCCTT GGAGTGGCGAAGTGA
mutB sequence (comprised in the mmcE-mutA-mutB construct shown in FIG. 15B and FIG. 16)	ATGTCTACCTTACCTCGCTTTGACAGTGTTGAT TTAGGAAATGCGCCGGTCCCAGCAGATGCTGC ACGTCGTTTTGAGGAACTTGCGGCGAAAGCCG GGACCGGCGAAGCCTGGGAAACTGCGGAACA AATTCCAGTAGGCACGTTGTTTAATGAAGACG TATACAAGGACATGGATTGGCTTGATACTTAC GCTGGCATTCCTCCCTTCGTCCATGGTCCGTA CGCTACTATGTATGCATTTCGTCCTTGGACCA TTCGCCAATATGCCGGTTTTTCGACTGCAAAG GAGTCAAACGCATTTTACCGTCGTAATTTGGC TGCAGGCCAGAAAGGTCTTAGTGTTGCTTTTG ACTTACCCACTCACCGCGGTTATGATTCCGAC AACCCCCGCGTGGCCGGAGATGTTGGTATGGC CGGTGTGGCTATCGATTCGATTTATGACATGC GTGAGCTGTTCGCCGGCATCCCATTAGATCAG ATGAGCGTGTCGATGACAATGAACGGTGCTGT CTTGCCGATTTTGGCTCTTTATGTGGTTACGGC GGAGGAGCAAGGCGTGAAGCCAGAACAACTG GCGGGTACTATTCAAAATGATATTCTGAAGGA ATTTATGGTTCGTAATACATATATTTACCCGC CGCAACCTAGTATGCGCATTATCAGCGAGATT TTTGCATACACATCAGCAAACATGCCGAAGTG GAACTCCATTAGTATCAGCGGCTATCATATGC AGGAGGCTGGAGCGACTGCGGATATCGAGAT GGCGTATACCTTAGCTGATGGAGTTGATTACA TCCGTGCTGGTGAGTCAGTAGGACTTAATGTG GACCAATTTGCTCCACGCCTGTCCTTCTTCTGG GGCATTGGTATGAACTTTTTCATGGAGGTAGC GAAGTTACGCGCTGCCCGTATGCTGTGGGCGA AGCTTGTCCACCAGTTCGGCCCGAAAAACCCG AAGAGTATGTCTCTGCGCACGCACTCTCAAAC ATCGGGTTGGTCTTTGACAGCTCAAGACGTAT ATAATAACGTTGTACGTACATGCATCGAAGCC ATGGCTGCTACTCAAGGCCATACTCAATCACT	SEQ ID NO: 34

-210WO 2017/023818

PCT/US2016/044922

	TCATACAAATTCGTTGGATGAAGCCATTGCAT TGCCTACGGACTTTTCAGCCCGCATTGCCCGC AATACTCAATTATTTCTGCAACAAGAGAGCGG GACGACTCGTGTGATCGACCCTTGGTCAGGTT CCGCATACGTCGAAGAGTTGACTTGGGATTTA GCTCGTAAAGCCTGGGGGCATATTCAGGAGG TTGAGAAGGTGGGGGGCATGGCTAAGGCAAT CGAGAAGGGGATTCCGAAGATGCGCATTGAG GAGGCAGCCGCCCGTACCCAAGCACGTATTG ATTCGGGACGCCAGCCATTAATTGGGGTCAAT AAATACCGTCTGGAGCACGAACCACCCCTGG ATGTGTTGAAGGTAGACAATAGCACCGTGTTA GCTGAGCAAAAGGCCAAACTTGTTAAATTGC GCGCAGAACGCGACCCAGAAAAGGTCAAGGC TGCTCTGGACAAAATCACTTGGGCGGCTGGCA ATCCTGATGATAAAGACCCTGATCGCAACTTA TTAAAGCTGTGCATTGATGCGGGGCGCGCGAT GGCAACGGTAGGAGAGATGAGTGACGCTTTA GAGAAAGTTTTTGGGCGCTACACAGCGCAAA TTCGCACTATTTCAGGAGTATATTCAAAAGAA GTCAAAAACACTCCGGAAGTCGAGGAGGCTC GCGAACTGGTAGAAGAGTTTGAGCAGGCCGA AGGCCGTCGCCCACGTATCCTGCTGGCTAAAA TGGGGCAGGACGGTCATGACCGTGGGCAAAA GGTCATCGCGACTGCATACGCCGATTTGGGAT TTGACGTGGACGTTGGCCCGTTATTCCAAACT CCCGAGGAAACTGCTCGCCAAGCCGTCGAAG CCGATGTGCACGTAGTGGGGGTGAGCTCTCTG GCGGGAGGGCATCTTACGCTTGTGCCTGCGCT TCGCAAAGAGCTGGACAAGTTGGGTCGTCCA GATATTCTGATTACCGTAGGAGGGGTTATTCC CGAGCAGGACTTCGATGAGCTTCGTAAGGAT GGCGCTGTTGAAATCTACACACCGGGGACGG TCATTCCAGAATCGGCTATCTCTTTAGTTAAA AAATTGCGCGCCTCCCTGGATGCT
Construct comprising TetR (reverse orientation, lowercase) and prpE-accApccB gene cassette driven by tet inducible promoter (italics) (as shown in FIG. 15B and FIG. 16); ribosome binding sites are underlined ;coding sequences bold and underlined	Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaa ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagc ttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttctttag cgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccaca gcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaa ggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaat gtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgtt attacgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgagtat ggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattt tttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagttta cgggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgtta atcac tttact111ate taatctagacatcatTAA TTC CTAA ΊΊΊΤΊG TTGACACTCTATCATTGATAGAGTTATTTTACCAC	SEQ ID NO: 35

-211WO 2017/023818

PCT/US2016/044922

	TCCCTATCAGTGATAGAGAAAAGTGAAAAAGGCG TAAGTTCAACAGGAGAGCATTXAAGGCGAAAGA TCAACAGGAGAGCATTATGTCTTTTAGCGAA
TTTTATCAGCGTTCGATTAACGAACCGGAG
AAGTTCTGGGCCGAGCAGGCCCGGCGTATT
GACTGGCAGACGCCCTTTACGCAAACGCTC
GACCACAGCAACCCGCCGTTTGCCCGTTGG
TTTTGTGAAGGCCGAACCAACTTGTGTCAC
AACGCTATCGACCGCTGGCTGGAGAAACAG
CCAGAGGCGCTGGCATTGATTGCCGTCTCT
TCGGAAACAGAGGAAGAGCGTACCTTTACC
TTCCGCCAGTTACATGACGAAGTGAATGCG
GTGGCGTCAATGCTGCGCTCACTGGGCGTG
CAGCGTGGCGATCGGGTGCTGGTGTATATG
CCGATGATTGCCGAAGCGCATATTACCCTG
CTGGCCTGCGCGCGCATTGGTGCTATTCAC
TCGGTGGTGTTTGGGGGATTTGCTTCGCAC
AGCGTGGCAACGCGAATTGATGACGCTAAA
CCGGTGCTGATTGTCTCGGCTGATGCCGGG
GCGCGCGGCGGTAAAATCATTCCGTATAAA
AAATTGCTCGACGATGCGATAAGTCAGGCA
CAGCATCAGCCGCGTCACGTTTTACTGGTG
GATCGCGGGCTGGCGAAAATGGCGCGCGT
TAGCGGGCGGGATGTCGATTTCGCGTCGTT
GCGCCATCAACACATCGGCGCGCGGGTGC
CGGTGGCATGGCTGGAATCCAACGAAACCT
CCTGCATTCTCTACACCTCCGGCACGACCG
GCAAACCTAAAGGTGTGCAGCGTGATGTCG
GCGGATATGCGGTGGCGCTGGCGACCTCG
ATGGACACCATTTTTGGCGGCAAAGCGGGC
GGCGTGTTCTTTTGTGCTTCGGATATCGGC
TGGGTGGTAGGGCATTCGTATATCGTTTAC
GCGCCGCTGCTGGCGGGGATGGCGACTAT
CGTTTACGAAGGATTGCCGACCTGGCCGGA
CTGCGGCGTGTGGTGGAAAATTGTCGAGAA
ATATCAGGTTAGCCGCATGTTCTCAGCGCC
GACCGCCATTCGCGTGCTGAAAAAATTCCC
TACCGCTGAAATTCGCAAACACGATCTTTC
GTCGCTGGAAGTGCTCTATCTGGCTGGAGA
ACCGCTGGACGAGCCGACCGCCAGTTGGG
TGAGCAATACGCTGGATGTGCCGGTCATCG
ACAACTACTGGCAGACCGAATCCGGCTGGC
CGATTATGGCGATTGCTCGCGGTCTGGATG
ACAGACCGACGCGTCTGGGAAGCCCCGGC
GTGCCGATGTATGGCTATAACGTGCAGTTG
CTCAATGAAGTCACCGGCGAACCGTGTGGC
GTCAATGAGAAAGGGATGCTGGTAGTGGA
GGGGCCATTGCCGCCAGGCTGTATTCAAAC
CATCTGGGGCGACGACGACCGCTTTGTGAA

-212WO 2017/023818

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	GACGTACTGGTCGCTGTTTTCCCGTCCGGT
GTACGCCACTTTTGACTGGGGCATCCGCGA
TGCTGACGGTTATCACTTTATTCTCGGGCG
CACTGACGATGTGATTAACGTTGCCGGACA
TCGGCTGGGTACGCGTGAGATTGAAGAGA
GTATCTCCAGTCATCCGGGCGTTGCCGAAG
TGGCGGTGGTTGGGGTGAAAGATGCGCTG
AAAGGGCAGGTGGCGGTGGCGTTTGTCATT
CCGAAAGAGAGCGACAGTCTGGAAGACCG
TGAGGTGGCGCACTCGCAAGAGAAGGCGA
TTATGGCGCTGGTGGACAGCCAGATTGGCA
ACTTTGGCCGCCCGGCGCACGTCTGGTTTG
TCTCGCAATTGCCAAAAACGCGATCCGGAA
AAATGCTGCGCCGCACGATCCAGGCGATTT
GCGAAGGACGCGATCCTGGGGATCTGACG
ACCATTGATGATCCGGCGTCGTTGGATCAG
ATCCGCCAGGCGATGGAAGAGTAGTACTAG ATTCAATATAGAGTAAAAGAGGTAAGAGTAT
CCATGCGTAAAGTTCTGATCGCTAATCGTG GAGAAATTGCTGTACGTGTAGCACGTGCAT
GTCGTGATGCGGGAATCGCATCAGTAGCCG
TATACGCGGACCCGGATCGTGACGCGTTGC
ATGTGCGCGCGGCGGACGAAGCATTTGCA
CTGGGTGGTGATACGCCTGCAACATCTTAC
TTAGACATCGCCAAGGTGTTAAAGGCTGCA
CGTGAGAGTGGTGCAGACGCCATTCATCCC
GGTTACGGCTTTTTAAGTGAAAATGCCGAG
TTCGCGCAGGCCGTGTTAGATGCGGGTCTT
ATCTGGATCGGACCACCGCCCCATGCAATC
CGCGATCGTGGGGAAAAAGTTGCAGCTCG
CCATATTGCCCAGCGTGCTGGGGCGCCGCT
GGTTGCGGGCACCCCTGACCCGGTTTCTGG
TGCTGACGAAGTCGTCGCCTTCGCGAAAGA
GCATGGACTGCCGATCGCGATTAAGGCTGC
TTTTGGAGGCGGTGGTCGTGGTTTAAAGGT
TGCCCGTACATTGGAAGAAGTGCCCGAGTT
ATATGACTCCGCCGTGCGTGAAGCTGTGGC
GGCATTCGGACGTGGCGAATGTTTCGTGGA
GCGCTATTTAGACAAACCGCGTCATGTAGA
AACCCAGTGCTTGGCAGATACTCACGGTAA
TGTAGTTGTGGTTTCTACTCGCGACTGTTC
GTTACAGCGTCGTCATCAGAAACTGGTAGA
GGAGGCACCCGCCCCGTTTTTAAGCGAAGC
TCAGACAGAGCAACTGTACTCCTCCTCCAA
GGCTATTCTTAAGGAAGCTGGGTATGGTGG
AGCGGGAACCGTTGAGTTTTTAGTAGGTAT
GGATGGTACTATCTTCTTCTTGGAGGTCAA
TACCCGCCTGCAGGTGGAGCACCCTGTGAC
CGAAGAAGTCGCAGGGATCGACCTGGTCC

-213WO 2017/023818

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	GTGAAATGTTCCGCATTGCAGATGGCGAGG
AGCTGGGGTACGACGATCCAGCCCTTCGCG
GCCACTCGTTCGAATTTCGCATCAATGGGG
AGGACCCAGGTCGTGGTTTTTTGCCCGCAC
CTGGTACGGTTACGCTTTTTGATGCTCCGA
CCGGACCCGGAGTCCGCCTGGATGCCGGG
GTTGAGTCAGGTTCCGTAATCGGACCGGCA
TGGGACTCACTGCTGGCTAAACTTATCGTT
ACCGGGCGTACACGTGCCGAGGCGCTTCA
GCGCGCAGCCCGCGCCTTAGATGAATTTAC
GGTTGAGGGCATGGCAACCGCGATCCCTTT
CCATCGCACAGTAGTACGCGATCCAGCATT
CGCTCCTGAGCTTACCGGGTCAACGGACCC
ATTCACCGTTCATACACGCTGGATTGAAAC
TGAATTTGTCAACGAAATTAAGCCTTTTAC
CACCCCTGCCGACACGGAGACAGATGAAG
AGTCTGGGCGCGAGACAGTGGTAGTCGAG
GTCGGTGGGAAACGCTTAGAGGTAAGTCTT
CCGTCCAGCCTGGGAATGTCGTTGGCCCGT
ACCGGCCTTGCCGCGGGGGCCCGCCCCAA
ACGCCGCGCGGCCAAGAAGTCAGGCCCTG
CAGCATCGGGTGATACACTGGCATCTCCTA
TGCAAGGTACGATCGTAAAGATCGCCGTGG
AAGAGGGACAAGAAGTACAGGAGGGAGATCT GATTGTGGTTCTTGAAGCTATGAAGATGGAAC AGCCACTTAATGCCCACCGTTCGGGAACCATT AAGGGGCTTACTGCTGAAGTAGGTGCTTCACT GACGTCGGGCGCCGCTATCTGTGAAATCAAG GATTGATAACGCTAACGAAAAAGTTAAATAC AGGAACAAGAGAACATATGTCGGAGCCCGA
GGAACAGCAGCCAGATATCCACACGACAGC
GGGCAAGTTAGCTGATCTTCGTCGCCGCAT
CGAAGAGGCAACGCACGCCGGTTCTGCGC
GCGCGGTGGAGAAACAGCACGCGAAGGGT
AAACTTACGGCTCGTGAGCGTATCGATTTG
TTGCTGGACGAAGGGTCTTTTGTAGAGCTT
GATGAGTTTGCGCGTCACCGTTCGACGAAT
TTCGGACTGGATGCCAACCGTCCATATGGA
GATGGAGTGGTGACTGGCTATGGAACTGTT
GACGGACGTCCGGTTGCCGTCTTTTCGCAA
GACTTTACGGTCTTTGGGGGCGCTCTGGGG
GAAGTATACGGGCAAAAAATTGTGAAGGTC
ATGGATTTCGCTCTTAAGACCGGGTGTCCC
GTCGTGGGTATTAATGACTCAGGTGGGGCA
CGCATTCAAGAGGGTGTAGCAAGTCTGGGC
GCGTATGGAGAGATTTTCCGTCGCAATACG
CACGCGTCGGGCGTGATCCCTCAGATTTCG
CTTGTAGTTGGCCCATGCGCAGGGGGAGCT
GTGTACTCTCCAGCTATTACTGACTTTACG

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PCT/US2016/044922

	GTAATGGTCGACCAAACATCGCATATGTTT
ATCACCGGACCCGATGTGATTAAGACAGTG
ACAGGGGAGGATGTGGGTTTTGAGGAACTT
GGTGGTGCGCGTACGCACAACAGTACGTCT
GGGGTTGCCCATCATATGGCTGGGGATGA
GAAAGACGCTGTGGAGTATGTTAAGCAATT
ATTGAGTTATTTGCCGTCGAACAATTTAAG
TGAGCCTCCGGCGTTTCCTGAAGAGGCTGA
TTTAGCCGTTACGGACGAAGATGCGGAATT
AGATACAATTGTGCCGGATTCGGCTAACCA
ACCCTATGATATGCATTCTGTAATCGAGCA
TGTCCTTGACGATGCGGAATTTTTCGAGAC
TCAACCGTTGTTTGCCCCCAACATCCTGAC
CGGCTTTGGTCGCGTTGAAGGCCGTCCGGT
GGGTATCGTGGCGAATCAGCCGATGCAGTT
TGCTGGATGCTTAGATATCACTGCCTCAGA
AAAAGCTGCTCGTTTCGTTCGCACTTGCGA
CGCTTTCAACGTCCCTGTGCTTACGTTTGT
AGACGTCCCCGGGTTTTTACCGGGCGTAGA
TCAGGAGCATGACGGGATCATCCGCCGCG
GTGCGAAGTTGATTTTTGCCTATGCAGAAG
CGACCGTGCCGTTGATCACAGTAATCACGC
GCAAAGCCTTCGGAGGTGCGTATGACGTAA
TGGGCTCAAAACACCTTGGCGCTGACCTTA
ATCTGGCATGGCCCACGGCCCAAATCGCTG
TAATGGGCGCTCAAGGTGCTGTAAACATCC
TTCATCGTCGTACGATTGCAGATGCGGGGG
ACGATGCGGAAGCCACGCGCGCCCGTTTAA
TTCAAGAGTACGAGGATGCTTTATTAAATC
CCTATACTGCGGCTGAGCGCGGGTATGTAG
ACGCGGTCATCATGCCCTCAGATACTCGCC
GTCATATCGTACGTGGTTTACGCCAATTAC
GCACCAAGCGCGAGTCTTTACCCCCGAAAA
AGCACGGGAACATTCCCCTT TGAGGAGGTCGGATAAGGCGCTCGCGCCGCA TCCGACACCGTGCGCAGATGCCTGATGCGACG CTGACGCGTCTTATCATGCCTCGCTCTCGAGT CCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT TACAACCAATTAACCAATTCTGAT
Construct comprising a prpE-accA-pccB gene cassette under the control of the Ptet promoter (as shown in FIG. 15B and FIG. 16) ribosome binding sites are underlined L3S2P11 terminator in italics; his terminator in	GAGTTATTTTACCACTCCCTATCAGTGATAGAGAA AAGTGAATAAGGCGTAAGTTCAACAGGAGAGCAT 7TAAGGCGTAAGTTCAACAGGAGAGCATTAT GTCTTTTAGCGAATTTTATCAGCGTTCGATT	SEQ ID NO: 36
AACGAACCGGAGAAGTTCTGGGCCGAGCA GGCCCGGCGTATTGACTGGCAGACGCCCTT
TACGCAAACGCTCGACCACAGCAACCCGCC
GTTTGCCCGTTGGTTTTGTGAAGGCCGAAC

-215WO 2017/023818

PCT/US2016/044922

bold ; coding sequences bold and underlined	CAACTTGTGTCACAACGCTATCGACCGCTG
GCTGGAGAAACAGCCAGAGGCGCTGGCAT
TGATTGCCGTCTCTTCGGAAACAGAGGAAG
AGCGTACCTTTACCTTCCGCCAGTTACATG
ACGAAGTGAATGCGGTGGCGTCAATGCTGC
GCTCACTGGGCGTGCAGCGTGGCGATCGG
GTGCTGGTGTATATGCCGATGATTGCCGAA
GCGCATATTACCCTGCTGGCCTGCGCGCGC
ATTGGTGCTATTCACTCGGTGGTGTTTGGG
GGATTTGCTTCGCACAGCGTGGCAACGCGA
ATTGATGACGCTAAACCGGTGCTGATTGTC
TCGGCTGATGCCGGGGCGCGCGGCGGTAA
AATCATTCCGTATAAAAAATTGCTCGACGA
TGCGATAAGTCAGGCACAGCATCAGCCGCG
TCACGTTTTACTGGTGGATCGCGGGCTGGC
GAAAATGGCGCGCGTTAGCGGGCGGGATG
TCGATTTCGCGTCGTTGCGCCATCAACACA
TCGGCGCGCGGGTGCCGGTGGCATGGCTG
GAATCCAACGAAACCTCCTGCATTCTCTAC
ACCTCCGGCACGACCGGCAAACCTAAAGGT
GTGCAGCGTGATGTCGGCGGATATGCGGT
GGCGCTGGCGACCTCGATGGACACCATTTT
TGGCGGCAAAGCGGGCGGCGTGTTCTTTTG
TGCTTCGGATATCGGCTGGGTGGTAGGGCA
TTCGTATATCGTTTACGCGCCGCTGCTGGC
GGGGATGGCGACTATCGTTTACGAAGGATT
GCCGACCTGGCCGGACTGCGGCGTGTGGT
GGAAAATTGTCGAGAAATATCAGGTTAGCC
GCATGTTCTCAGCGCCGACCGCCATTCGCG
TGCTGAAAAAATTCCCTACCGCTGAAATTC
GCAAACACGATCTTTCGTCGCTGGAAGTGC
TCTATCTGGCTGGAGAACCGCTGGACGAGC
CGACCGCCAGTTGGGTGAGCAATACGCTG
GATGTGCCGGTCATCGACAACTACTGGCAG
ACCGAATCCGGCTGGCCGATTATGGCGATT
GCTCGCGGTCTGGATGACAGACCGACGCG
TCTGGGAAGCCCCGGCGTGCCGATGTATG
GCTATAACGTGCAGTTGCTCAATGAAGTCA
CCGGCGAACCGTGTGGCGTCAATGAGAAA
GGGATGCTGGTAGTGGAGGGGCCATTGCC
GCCAGGCTGTATTCAAACCATCTGGGGCGA
CGACGACCGCTTTGTGAAGACGTACTGGTC
GCTGTTTTCCCGTCCGGTGTACGCCACTTT
TGACTGGGGCATCCGCGATGCTGACGGTTA
TCACTTTATTCTCGGGCGCACTGACGATGT
GATTAACGTTGCCGGACATCGGCTGGGTAC
GCGTGAGATTGAAGAGAGTATCTCCAGTCA
TCCGGGCGTTGCCGAAGTGGCGGTGGTTG
GGGTGAAAGATGCGCTGAAAGGGCAGGTG

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PCT/US2016/044922

	GCGGTGGCGTTTGTCATTCCGAAAGAGAGC
GACAGTCTGGAAGACCGTGAGGTGGCGCA
CTCGCAAGAGAAGGCGATTATGGCGCTGGT
GGACAGCCAGATTGGCAACTTTGGCCGCCC
GGCGCACGTCTGGTTTGTCTCGCAATTGCC
AAAAACGCGATCCGGAAAAATGCTGCGCCG
CACGATCCAGGCGATTTGCGAAGGACGCG
ATCCTGGGGATCTGACGACCATTGATGATC
CGGCGTCGTTGGATCAGATCCGCCAGGCG
ATGGAAGAGTAGTACTAGATTCAATATAGAG
TAAAAGAGGTAAGAGTATCCATGCGTAAAGT
TCTGATCGCTAATCGTGGAGAAATTGCTGT
ACGTGTAGCACGTGCATGTCGTGATGCGGG
AATCGCATCAGTAGCCGTATACGCGGACCC
GGATCGTGACGCGTTGCATGTGCGCGCGG
CGGACGAAGCATTTGCACTGGGTGGTGATA
CGCCTGCAACATCTTACTTAGACATCGCCA
AGGTGTTAAAGGCTGCACGTGAGAGTGGT
GCAGACGCCATTCATCCCGGTTACGGCTTT
TTAAGTGAAAATGCCGAGTTCGCGCAGGCC
GTGTTAGATGCGGGTCTTATCTGGATCGGA
CCACCGCCCCATGCAATCCGCGATCGTGGG
GAAAAAGTTGCAGCTCGCCATATTGCCCAG
CGTGCTGGGGCGCCGCTGGTTGCGGGCAC
CCCTGACCCGGTTTCTGGTGCTGACGAAGT
CGTCGCCTTCGCGAAAGAGCATGGACTGCC
GATCGCGATTAAGGCTGCTTTTGGAGGCGG
TGGTCGTGGTTTAAAGGTTGCCCGTACATT
GGAAGAAGTGCCCGAGTTATATGACTCCGC
CGTGCGTGAAGCTGTGGCGGCATTCGGAC
GTGGCGAATGTTTCGTGGAGCGCTATTTAG
ACAAACCGCGTCATGTAGAAACCCAGTGCT
TGGCAGATACTCACGGTAATGTAGTTGTGG
TTTCTACTCGCGACTGTTCGTTACAGCGTC
GTCATCAGAAACTGGTAGAGGAGGCACCC
GCCCCGTTTTTAAGCGAAGCTCAGACAGAG
CAACTGTACTCCTCCTCCAAGGCTATTCTT
AAGGAAGCTGGGTATGGTGGAGCGGGAAC
CGTTGAGTTTTTAGTAGGTATGGATGGTAC
TATCTTCTTCTTGGAGGTCAATACCCGCCT
GCAGGTGGAGCACCCTGTGACCGAAGAAG
TCGCAGGGATCGACCTGGTCCGTGAAATGT
TCCGCATTGCAGATGGCGAGGAGCTGGGG
TACGACGATCCAGCCCTTCGCGGCCACTCG
TTCGAATTTCGCATCAATGGGGAGGACCCA
GGTCGTGGTTTTTTGCCCGCACCTGGTACG
GTTACGCTTTTTGATGCTCCGACCGGACCC
GGAGTCCGCCTGGATGCCGGGGTTGAGTC
AGGTTCCGTAATCGGACCGGCATGGGACTC

-217WO 2017/023818

PCT/US2016/044922

	ACTGCTGGCTAAACTTATCGTTACCGGGCG
TACACGTGCCGAGGCGCTTCAGCGCGCAG
CCCGCGCCTTAGATGAATTTACGGTTGAGG
GCATGGCAACCGCGATCCCTTTCCATCGCA
CAGTAGTACGCGATCCAGCATTCGCTCCTG
AGCTTACCGGGTCAACGGACCCATTCACCG
TTCATACACGCTGGATTGAAACTGAATTTG
TCAACGAAATTAAGCCTTTTACCACCCCTG
CCGACACGGAGACAGATGAAGAGTCTGGG
CGCGAGACAGTGGTAGTCGAGGTCGGTGG
GAAACGCTTAGAGGTAAGTCTTCCGTCCAG
CCTGGGAATGTCGTTGGCCCGTACCGGCCT
TGCCGCGGGGGCCCGCCCCAAACGCCGCG
CGGCCAAGAAGTCAGGCCCTGCAGCATCG
GGTGATACACTGGCATCTCCTATGCAAGGT
ACGATCGTAAAGATCGCCGTGGAAGAGGGA
CAAGAAGTACAGGAGGGAGATCTGATTGTGG TTCTTGAAGCTATGAAGATGGAACAGCCACTT AATGCCCACCGTTCGGGAACCATTAAGGGGCT TACTGCTGAAGTAGGTGCTTCACTGACGTCGG GCGCCGCTATCTGTGAAATCAAGGATTGATAA CGCTAACGAAAAAGTTAAATACAGGAACAAG AGAACATATGTCGGAGCCCGAGGAACAGCA
GCCAGATATCCACACGACAGCGGGCAAGTT
AGCTGATCTTCGTCGCCGCATCGAAGAGGC
AACGCACGCCGGTTCTGCGCGCGCGGTGG
AGAAACAGCACGCGAAGGGTAAACTTACG
GCTCGTGAGCGTATCGATTTGTTGCTGGAC
GAAGGGTCTTTTGTAGAGCTTGATGAGTTT
GCGCGTCACCGTTCGACGAATTTCGGACTG
GATGCCAACCGTCCATATGGAGATGGAGTG
GTGACTGGCTATGGAACTGTTGACGGACGT
CCGGTTGCCGTCTTTTCGCAAGACTTTACG
GTCTTTGGGGGCGCTCTGGGGGAAGTATAC
GGGCAAAAAATTGTGAAGGTCATGGATTTC
GCTCTTAAGACCGGGTGTCCCGTCGTGGGT
ATTAATGACTCAGGTGGGGCACGCATTCAA
GAGGGTGTAGCAAGTCTGGGCGCGTATGG
AGAGATTTTCCGTCGCAATACGCACGCGTC
GGGCGTGATCCCTCAGATTTCGCTTGTAGT
TGGCCCATGCGCAGGGGGAGCTGTGTACT
CTCCAGCTATTACTGACTTTACGGTAATGG
TCGACCAAACATCGCATATGTTTATCACCG
GACCCGATGTGATTAAGACAGTGACAGGG
GAGGATGTGGGTTTTGAGGAACTTGGTGGT
GCGCGTACGCACAACAGTACGTCTGGGGTT
GCCCATCATATGGCTGGGGATGAGAAAGAC
GCTGTGGAGTATGTTAAGCAATTATTGAGT
TATTTGCCGTCGAACAATTTAAGTGAGCCT

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PCT/US2016/044922

	CCGGCGTTTCCTGAAGAGGCTGATTTAGCC
GTTACGGACGAAGATGCGGAATTAGATACA
ATTGTGCCGGATTCGGCTAACCAACCCTAT
GATATGCATTCTGTAATCGAGCATGTCCTT
GACGATGCGGAATTTTTCGAGACTCAACCG
TTGTTTGCCCCCAACATCCTGACCGGCTTT
GGTCGCGTTGAAGGCCGTCCGGTGGGTAT
CGTGGCGAATCAGCCGATGCAGTTTGCTGG
ATGCTTAGATATCACTGCCTCAGAAAAAGC
TGCTCGTTTCGTTCGCACTTGCGACGCTTT
CAACGTCCCTGTGCTTACGTTTGTAGACGT
CCCCGGGTTTTTACCGGGCGTAGATCAGGA
GCATGACGGGATCATCCGCCGCGGTGCGA
AGTTGATTTTTGCCTATGCAGAAGCGACCG
TGCCGTTGATCACAGTAATCACGCGCAAAG
CCTTCGGAGGTGCGTATGACGTAATGGGCT
CAAAACACCTTGGCGCTGACCTTAATCTGG
CATGGCCCACGGCCCAAATCGCTGTAATGG
GCGCTCAAGGTGCTGTAAACATCCTTCATC
GTCGTACGATTGCAGATGCGGGGGACGAT
GCGGAAGCCACGCGCGCCCGTTTAATTCAA
GAGTACGAGGATGCTTTATTAAATCCCTAT
ACTGCGGCTGAGCGCGGGTATGTAGACGC
GGTCATCATGCCCTCAGATACTCGCCGTCA
TATCGTACGTGGTTTACGCCAATTACGCAC
CAAGCGCGAGTCTTTACCCCCGAAAAAGCA
CGGGAACATTCCCCTT
Construct comprising a prpE-accA-pccB gene cassette; (as shown in FIG. 15B and FIG. 16) ribosome binding sites are underlined; coding sequences bold and underlined	TAAGGCGTAAGTTCAACAGGAGAGCATTATG	SEQ ID NO: 37
TCTTTTAGCGAATTTTATCAGCGTTCGATTA ACGAACCGGAGAAGTTCTGGGCCGAGCAG
GCCCGGCGTATTGACTGGCAGACGCCCTTT
ACGCAAACGCTCGACCACAGCAACCCGCCG
TTTGCCCGTTGGTTTTGTGAAGGCCGAACC
AACTTGTGTCACAACGCTATCGACCGCTGG
CTGGAGAAACAGCCAGAGGCGCTGGCATT
GATTGCCGTCTCTTCGGAAACAGAGGAAGA
GCGTACCTTTACCTTCCGCCAGTTACATGA
CGAAGTGAATGCGGTGGCGTCAATGCTGC
GCTCACTGGGCGTGCAGCGTGGCGATCGG
GTGCTGGTGTATATGCCGATGATTGCCGAA
GCGCATATTACCCTGCTGGCCTGCGCGCGC
ATTGGTGCTATTCACTCGGTGGTGTTTGGG
GGATTTGCTTCGCACAGCGTGGCAACGCGA
ATTGATGACGCTAAACCGGTGCTGATTGTC
TCGGCTGATGCCGGGGCGCGCGGCGGTAA
AATCATTCCGTATAAAAAATTGCTCGACGA
TGCGATAAGTCAGGCACAGCATCAGCCGCG
TCACGTTTTACTGGTGGATCGCGGGCTGGC

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PCT/US2016/044922

	GAAAATGGCGCGCGTTAGCGGGCGGGATG
TCGATTTCGCGTCGTTGCGCCATCAACACA
TCGGCGCGCGGGTGCCGGTGGCATGGCTG
GAATCCAACGAAACCTCCTGCATTCTCTAC
ACCTCCGGCACGACCGGCAAACCTAAAGGT
GTGCAGCGTGATGTCGGCGGATATGCGGT
GGCGCTGGCGACCTCGATGGACACCATTTT
TGGCGGCAAAGCGGGCGGCGTGTTCTTTTG
TGCTTCGGATATCGGCTGGGTGGTAGGGCA
TTCGTATATCGTTTACGCGCCGCTGCTGGC
GGGGATGGCGACTATCGTTTACGAAGGATT
GCCGACCTGGCCGGACTGCGGCGTGTGGT
GGAAAATTGTCGAGAAATATCAGGTTAGCC
GCATGTTCTCAGCGCCGACCGCCATTCGCG
TGCTGAAAAAATTCCCTACCGCTGAAATTC
GCAAACACGATCTTTCGTCGCTGGAAGTGC
TCTATCTGGCTGGAGAACCGCTGGACGAGC
CGACCGCCAGTTGGGTGAGCAATACGCTG
GATGTGCCGGTCATCGACAACTACTGGCAG
ACCGAATCCGGCTGGCCGATTATGGCGATT
GCTCGCGGTCTGGATGACAGACCGACGCG
TCTGGGAAGCCCCGGCGTGCCGATGTATG
GCTATAACGTGCAGTTGCTCAATGAAGTCA
CCGGCGAACCGTGTGGCGTCAATGAGAAA
GGGATGCTGGTAGTGGAGGGGCCATTGCC
GCCAGGCTGTATTCAAACCATCTGGGGCGA
CGACGACCGCTTTGTGAAGACGTACTGGTC
GCTGTTTTCCCGTCCGGTGTACGCCACTTT
TGACTGGGGCATCCGCGATGCTGACGGTTA
TCACTTTATTCTCGGGCGCACTGACGATGT
GATTAACGTTGCCGGACATCGGCTGGGTAC
GCGTGAGATTGAAGAGAGTATCTCCAGTCA
TCCGGGCGTTGCCGAAGTGGCGGTGGTTG
GGGTGAAAGATGCGCTGAAAGGGCAGGTG
GCGGTGGCGTTTGTCATTCCGAAAGAGAGC
GACAGTCTGGAAGACCGTGAGGTGGCGCA
CTCGCAAGAGAAGGCGATTATGGCGCTGGT
GGACAGCCAGATTGGCAACTTTGGCCGCCC
GGCGCACGTCTGGTTTGTCTCGCAATTGCC
AAAAACGCGATCCGGAAAAATGCTGCGCCG
CACGATCCAGGCGATTTGCGAAGGACGCG
ATCCTGGGGATCTGACGACCATTGATGATC
CGGCGTCGTTGGATCAGATCCGCCAGGCG
ATGGAAGAGTAGTACTAGATTCAATATAGAG
TAAAAGAGGTAAGAGTATCCATGCGTAAAGT
TCTGATCGCTAATCGTGGAGAAATTGCTGT
ACGTGTAGCACGTGCATGTCGTGATGCGGG
AATCGCATCAGTAGCCGTATACGCGGACCC
GGATCGTGACGCGTTGCATGTGCGCGCGG

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	CGGACGAAGCATTTGCACTGGGTGGTGATA
CGCCTGCAACATCTTACTTAGACATCGCCA
AGGTGTTAAAGGCTGCACGTGAGAGTGGT
GCAGACGCCATTCATCCCGGTTACGGCTTT
TTAAGTGAAAATGCCGAGTTCGCGCAGGCC
GTGTTAGATGCGGGTCTTATCTGGATCGGA
CCACCGCCCCATGCAATCCGCGATCGTGGG
GAAAAAGTTGCAGCTCGCCATATTGCCCAG
CGTGCTGGGGCGCCGCTGGTTGCGGGCAC
CCCTGACCCGGTTTCTGGTGCTGACGAAGT
CGTCGCCTTCGCGAAAGAGCATGGACTGCC
GATCGCGATTAAGGCTGCTTTTGGAGGCGG
TGGTCGTGGTTTAAAGGTTGCCCGTACATT
GGAAGAAGTGCCCGAGTTATATGACTCCGC
CGTGCGTGAAGCTGTGGCGGCATTCGGAC
GTGGCGAATGTTTCGTGGAGCGCTATTTAG
ACAAACCGCGTCATGTAGAAACCCAGTGCT
TGGCAGATACTCACGGTAATGTAGTTGTGG
TTTCTACTCGCGACTGTTCGTTACAGCGTC
GTCATCAGAAACTGGTAGAGGAGGCACCC
GCCCCGTTTTTAAGCGAAGCTCAGACAGAG
CAACTGTACTCCTCCTCCAAGGCTATTCTT
AAGGAAGCTGGGTATGGTGGAGCGGGAAC
CGTTGAGTTTTTAGTAGGTATGGATGGTAC
TATCTTCTTCTTGGAGGTCAATACCCGCCT
GCAGGTGGAGCACCCTGTGACCGAAGAAG
TCGCAGGGATCGACCTGGTCCGTGAAATGT
TCCGCATTGCAGATGGCGAGGAGCTGGGG
TACGACGATCCAGCCCTTCGCGGCCACTCG
TTCGAATTTCGCATCAATGGGGAGGACCCA
GGTCGTGGTTTTTTGCCCGCACCTGGTACG
GTTACGCTTTTTGATGCTCCGACCGGACCC
GGAGTCCGCCTGGATGCCGGGGTTGAGTC
AGGTTCCGTAATCGGACCGGCATGGGACTC
ACTGCTGGCTAAACTTATCGTTACCGGGCG
TACACGTGCCGAGGCGCTTCAGCGCGCAG
CCCGCGCCTTAGATGAATTTACGGTTGAGG
GCATGGCAACCGCGATCCCTTTCCATCGCA
CAGTAGTACGCGATCCAGCATTCGCTCCTG
AGCTTACCGGGTCAACGGACCCATTCACCG
TTCATACACGCTGGATTGAAACTGAATTTG
TCAACGAAATTAAGCCTTTTACCACCCCTG
CCGACACGGAGACAGATGAAGAGTCTGGG
CGCGAGACAGTGGTAGTCGAGGTCGGTGG
GAAACGCTTAGAGGTAAGTCTTCCGTCCAG
CCTGGGAATGTCGTTGGCCCGTACCGGCCT
TGCCGCGGGGGCCCGCCCCAAACGCCGCG
CGGCCAAGAAGTCAGGCCCTGCAGCATCG
GGTGATACACTGGCATCTCCTATGCAAGGT

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	ACGATCGTAAAGATCGCCGTGGA AGAGGGACAAGAAGTACAGGAGGGAGATCTG ATTGTGGTTCTTGAAGCTATGAAGATGGAACA GCCACTTAATGCCCACCGTTCGGGAACCATTA AGGGGCTTACTGCTGAAGTAGGTGCTTCACTG ACGTCGGGCGCCGCTATCTGTGAAATCAAGG ATTGATAACGCTAACGAAAAAGTTAAATACA GGAACAAGAGAACATATGTCGGAGCCCGAG
GAACAGCAGCCAGATATCCACACGACAGCG
GGCAAGTTAGCTGATCTTCGTCGCCGCATC
GAAGAGGCAACGCACGCCGGTTCTGCGCG
CGCGGTGGAGAAACAGCACGCGAAGGGTA
AACTTACGGCTCGTGAGCGTATCGATTTGT
TGCTGGACGAAGGGTCTTTTGTAGAGCTTG
ATGAGTTTGCGCGTCACCGTTCGACGAATT
TCGGACTGGATGCCAACCGTCCATATGGAG
ATGGAGTGGTGACTGGCTATGGAACTGTTG
ACGGACGTCCGGTTGCCGTCTTTTCGCAAG
ACTTTACGGTCTTTGGGGGCGCTCTGGGGG
AAGTATACGGGCAAAAAATTGTGAAGGTCA
TGGATTTCGCTCTTAAGACCGGGTGTCCCG
TCGTGGGTATTAATGACTCAGGTGGGGCAC
GCATTCAAGAGGGTGTAGCAAGTCTGGGC
GCGTATGGAGAGATTTTCCGTCGCAATACG
CACGCGTCGGGCGTGATCCCTCAGATTTCG
CTTGTAGTTGGCCCATGCGCAGGGGGAGCT
GTGTACTCTCCAGCTATTACTGACTTTACG
GTAATGGTCGACCAAACATCGCATATGTTT
ATCACCGGACCCGATGTGATTAAGACAGTG
ACAGGGGAGGATGTGGGTTTTGAGGAACTT
GGTGGTGCGCGTACGCACAACAGTACGTCT
GGGGTTGCCCATCATATGGCTGGGGATGA
GAAAGACGCTGTGGAGTATGTTAAGCAATT
ATTGAGTTATTTGCCGTCGAACAATTTAAG
TGAGCCTCCGGCGTTTCCTGAAGAGGCTGA
TTTAGCCGTTACGGACGAAGATGCGGAATT
AGATACAATTGTGCCGGATTCGGCTAACCA
ACCCTATGATATGCATTCTGTAATCGAGCA
TGTCCTTGACGATGCGGAATTTTTCGAGAC
TCAACCGTTGTTTGCCCCCAACATCCTGAC
CGGCTTTGGTCGCGTTGAAGGCCGTCCGGT
GGGTATCGTGGCGAATCAGCCGATGCAGTT
TGCTGGATGCTTAGATATCACTGCCTCAGA
AAAAGCTGCTCGTTTCGTTCGCACTTGCGA
CGCTTTCAACGTCCCTGTGCTTACGTTTGT
AGACGTCCCCGGGTTTTTACCGGGCGTAGA
TCAGGAGCATGACGGGATCATCCGCCGCG
GTGCGAAGTTGATTTTTGCCTATGCAGAAG
CGACCGTGCCGTTGATCACAGTAATCACGC

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	GCAAAGCCTTCGGAGGTGCGTATGACGTAA
TGGGCTCAAAACACCTTGGCGCTGACCTTA
ATCTGGCATGGCCCACGGCCCAAATCGCTG
TAATGGGCGCTCAAGGTGCTGTAAACATCC
TTCATCGTCGTACGATTGCAGATGCGGGGG
ACGATGCGGAAGCCACGCGCGCCCGTTTAA
TTCAAGAGTACGAGGATGCTTTATTAAATC
CCTATACTGCGGCTGAGCGCGGGTATGTAG
ACGCGGTCATCATGCCCTCAGATACTCGCC
GTCATATCGTACGTGGTTTACGCCAATTAC
GCACCAAGCGCGAGTCTTTACCCCCGAAAA
AGCACGGGAACATTCCCCTT
prpE sequence (comprised in the prpE-accA-pccB construct shown in FIG. 15B and FIG. 16)	ATGTCTTTTAGCGAATTTTATCAGCGTTCGATT AACGAACCGGAGAAGTTCTGGGCCGAGCAGG CCCGGCGTATTGACTGGCAGACGCCCTTTACG CAAACGCTCGACCACAGCAACCCGCCGTTTGC CCGTTGGTTTTGTGAAGGCCGAACCAACTTGT GTCACAACGCTATCGACCGCTGGCTGGAGAA ACAGCCAGAGGCGCTGGCATTGATTGCCGTCT CTTCGGAAACAGAGGAAGAGCGTACCTTTAC CTTCCGCCAGTTACATGACGAAGTGAATGCGG TGGCGTCAATGCTGCGCTCACTGGGCGTGCAG CGTGGCGATCGGGTGCTGGTGTATATGCCGAT GATTGCCGAAGCGCATATTACCCTGCTGGCCT GCGCGCGCATTGGTGCTATTCACTCGGTGGTG TTTGGGGGATTTGCTTCGCACAGCGTGGCAAC GCGAATTGATGACGCTAAACCGGTGCTGATTG TCTCGGCTGATGCCGGGGCGCGCGGCGGTAA AATCATTCCGTATAAAAAATTGCTCGACGATG CGATAAGTCAGGCACAGCATCAGCCGCGTCA CGTTTTACTGGTGGATCGCGGGCTGGCGAAAA TGGCGCGCGTTAGCGGGCGGGATGTCGATTTC GCGTCGTTGCGCCATCAACACATCGGCGCGCG GGTGCCGGTGGCATGGCTGGAATCCAACGAA ACCTCCTGCATTCTCTACACCTCCGGCACGAC CGGCAAACCTAAAGGTGTGCAGCGTGATGTC GGCGGATATGCGGTGGCGCTGGCGACCTCGA TGGACACCATTTTTGGCGGCAAAGCGGGCGG CGTGTTCTTTTGTGCTTCGGATATCGGCTGGGT GGTAGGGCATTCGTATATCGTTTACGCGCCGC TGCTGGCGGGGATGGCGACTATCGTTTACGAA GGATTGCCGACCTGGCCGGACTGCGGCGTGTG GTGGAAAATTGTCGAGAAATATCAGGTTAGC CGCATGTTCTCAGCGCCGACCGCCATTCGCGT GCTGAAAAAATTCCCTACCGCTGAAATTCGCA AACACGATCTTTCGTCGCTGGAAGTGCTCTAT CTGGCTGGAGAACCGCTGGACGAGCCGACCG CCAGTTGGGTGAGCAATACGCTGGATGTGCCG	SEQ ID NO: 25

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	GTCATCGACAACTACTGGCAGACCGAATCCG GCTGGCCGATTATGGCGATTGCTCGCGGTCTG GATGACAGACCGACGCGTCTGGGAAGCCCCG GCGTGCCGATGTATGGCTATAACGTGCAGTTG CTCAATGAAGTCACCGGCGAACCGTGTGGCGT CAATGAGAAAGGGATGCTGGTAGTGGAGGGG CCATTGCCGCCAGGCTGTATTCAAACCATCTG GGGCGACGACGACCGCTTTGTGAAGACGTAC TGGTCGCTGTTTTCCCGTCCGGTGTACGCCAC TTTTGACTGGGGCATCCGCGATGCTGACGGTT ATCACTTTATTCTCGGGCGCACTGACGATGTG ATTAACGTTGCCGGACATCGGCTGGGTACGCG TGAGATTGAAGAGAGTATCTCCAGTCATCCGG GCGTTGCCGAAGTGGCGGTGGTTGGGGTGAA AGATGCGCTGAAAGGGCAGGTGGCGGTGGCG TTTGTCATTCCGAAAGAGAGCGACAGTCTGGA AGACCGTGAGGTGGCGCACTCGCAAGAGAAG GCGATTATGGCGCTGGTGGACAGCCAGATTG GCAACTTTGGCCGCCCGGCGCACGTCTGGTTT GTCTCGCAATTGCCAAAAACGCGATCCGGAA AAATGCTGCGCCGCACGATCCAGGCGATTTGC GAAGGACGCGATCCTGGGGATCTGACGACCA TTGATGATCCGGCGTCGTTGGATCAGATCCGC CAGGCGATGGAAGAGTAG
accA sequence (comprised in the prpE-accA-pccB construct shown in FIG. 15B and FIG. 16)	ATGCGTAAAGTTCTGATCGCTAATCGTGGAGA AATTGCTGTACGTGTAGCACGTGCATGTCGTG ATGCGGGAATCGCATCAGTAGCCGTATACGC GGACCCGGATCGTGACGCGTTGCATGTGCGCG CGGCGGACGAAGCATTTGCACTGGGTGGTGA TACGCCTGCAACATCTTACTTAGACATCGCCA AGGTGTTAAAGGCTGCACGTGAGAGTGGTGC AGACGCCATTCATCCCGGTTACGGCTTTTTAA GTGAAAATGCCGAGTTCGCGCAGGCCGTGTTA GATGCGGGTCTTATCTGGATCGGACCACCGCC CCATGCAATCCGCGATCGTGGGGAAAAAGTT GCAGCTCGCCATATTGCCCAGCGTGCTGGGGC GCCGCTGGTTGCGGGCACCCCTGACCCGGTTT CTGGTGCTGACGAAGTCGTCGCCTTCGCGAAA GAGCATGGACTGCCGATCGCGATTAAGGCTG CTTTTGGAGGCGGTGGTCGTGGTTTAAAGGTT GCCCGTACATTGGAAGAAGTGCCCGAGTTATA TGACTCCGCCGTGCGTGAAGCTGTGGCGGCAT TCGGACGTGGCGAATGTTTCGTGGAGCGCTAT TTAGACAAACCGCGTCATGTAGAAACCCAGT GCTTGGCAGATACTCACGGTAATGTAGTTGTG GTTTCTACTCGCGACTGTTCGTTACAGCGTCG TCATCAGAAACTGGTAGAGGAGGCACCCGCC CCGTTTTTAAGCGAAGCTCAGACAGAGCAACT	SEQ ID NO: 38

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	GTACTCCTCCTCCAAGGCTATTCTTAAGGAAG CTGGGTATGGTGGAGCGGGAACCGTTGAGTTT TTAGTAGGTATGGATGGTACTATCTTCTTCTTG GAGGTCAATACCCGCCTGCAGGTGGAGCACC CTGTGACCGAAGAAGTCGCAGGGATCGACCT GGTCCGTGAAATGTTCCGCATTGCAGATGGCG AGGAGCTGGGGTACGACGATCCAGCCCTTCG CGGCCACTCGTTCGAATTTCGCATCAATGGGG AGGACCCAGGTCGTGGTTTTTTGCCCGCACCT GGTACGGTTACGCTTTTTGATGCTCCGACCGG ACCCGGAGTCCGCCTGGATGCCGGGGTTGAGT CAGGTTCCGTAATCGGACCGGCATGGGACTCA CTGCTGGCTAAACTTATCGTTACCGGGCGTAC ACGTGCCGAGGCGCTTCAGCGCGCAGCCCGC GCCTTAGATGAATTTACGGTTGAGGGCATGGC AACCGCGATCCCTTTCCATCGCACAGTAGTAC GCGATCCAGCATTCGCTCCTGAGCTTACCGGG TCAACGGACCCATTCACCGTTCATACACGCTG GATTGAAACTGAATTTGTCAACGAAATTAAGC CTTTTACCACCCCTGCCGACACGGAGACAGAT GAAGAGTCTGGGCGCGAGACAGTGGTAGTCG AGGTCGGTGGGAAACGCTTAGAGGTAAGTCT TCCGTCCAGCCTGGGAATGTCGTTGGCCCGTA CCGGCCTTGCCGCGGGGGCCCGCCCCAAACG CCGCGCGGCCAAGAAGTCAGGCCCTGCAGCA TCGGGTGATACACTGGCATCTCCTATGCAAGG TACGATCGTAAAGATCGCCGTGGAAGAGGGA CAAGAAGTACAGGAGGGAGATCTGATTGTGG TTCTTGAAGCTATGAAGATGGAACAGCCACTT AATGCCCACCGTTCGGGAACCATTAAGGGGCT TACTGCTGAAGTAGGTGCTTCACTGACGTCGG GCGCCGCTATCTGTGAAATCAAGGATTG
pccB sequence (comprised in the prpE-accA-pccB construct shown in FIG. 15B and FIG. 16)	ATGTCGGAGCCCGAGGAACAGCAGCCAGATA TCCACACGACAGCGGGCAAGTTAGCTGATCTT CGTCGCCGCATCGAAGAGGCAACGCACGCCG GTTCTGCGCGCGCGGTGGAGAAACAGCACGC GAAGGGTAAACTTACGGCTCGTGAGCGTATC GATTTGTTGCTGGACGAAGGGTCTTTTGTAGA GCTTGATGAGTTTGCGCGTCACCGTTCGACGA ATTTCGGACTGGATGCCAACCGTCCATATGGA GATGGAGTGGTGACTGGCTATGGAACTGTTGA CGGACGTCCGGTTGCCGTCTTTTCGCAAGACT TTACGGTCTTTGGGGGCGCTCTGGGGGAAGTA TACGGGCAAAAAATTGTGAAGGTCATGGATTT CGCTCTTAAGACCGGGTGTCCCGTCGTGGGTA TTAATGACTCAGGTGGGGCACGCATTCAAGA GGGTGTAGCAAGTCTGGGCGCGTATGGAGAG ATTTTCCGTCGCAATACGCACGCGTCGGGCGT	SEQ ID NO: 39

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GATCCCTCAGATTTCGCTTGTAGTTGGCCCAT GCGCAGGGGGAGCTGTGTACTCTCCAGCTATT ACTGACTTTACGGTAATGGTCGACCAAACATC GCATATGTTTATCACCGGACCCGATGTGATTA AGACAGTGACAGGGGAGGATGTGGGTTTTGA GGAACTTGGTGGTGCGCGTACGCACAACAGT ACGTCTGGGGTTGCCCATCATATGGCTGGGGA TGAGAAAGACGCTGTGGAGTATGTTAAGCAA TTATTGAGTTATTTGCCGTCGAACAATTTAAG TGAGCCTCCGGCGTTTCCTGAAGAGGCTGATT TAGCCGTTACGGACGAAGATGCGGAATTAGA TACAATTGTGCCGGATTCGGCTAACCAACCCT ATGATATGCATTCTGTAATCGAGCATGTCCTT GACGATGCGGAATTTTTCGAGACTCAACCGTT GTTTGCCCCCAACATCCTGACCGGCTTTGGTC GCGTTGAAGGCCGTCCGGTGGGTATCGTGGCG AATCAGCCGATGCAGTTTGCTGGATGCTTAGA TATCACTGCCTCAGAAAAAGCTGCTCGTTTCG TTCGCACTTGCGACGCTTTCAACGTCCCTGTG CTTACGTTTGTAGACGTCCCCGGGTTTTTACC GGGCGTAGATCAGGAGCATGACGGGATCATC CGCCGCGGTGCGAAGTTGATTTTTGCCTATGC AGAAGCGACCGTGCCGTTGATCACAGTAATC ACGCGCAAAGCCTTCGGAGGTGCGTATGACG TAATGGGCTCAAAACACCTTGGCGCTGACCTT AATCTGGCATGGCCCACGGCCCAAATCGCTGT AATGGGCGCTCAAGGTGCTGTAAACATCCTTC ATCGTCGTACGATTGCAGATGCGGGGGACGA TGCGGAAGCCACGCGCGCCCGTTTAATTCAAG AGTACGAGGATGCTTTATTAAATCCCTATACT GCGGCTGAGCGCGGGTATGTAGACGCGGTCA TCATGCCCTCAGATACTCGCCGTCATATCGTA CGTGGTTTACGCCAATTACGCACCAAGCGCGA GTCTTTACCCCCGAAAAAGCACGGGAACATTC CCCTTTG

[0524] In certain constructs, the prpE pccB,-accAl and mmcE-mutAB cassettes are operably linked to a FNR-responsive promoter, which may be is further fused to a strong ribosome binding site sequence. For efficient translation, a 15 base pair ribosome binding site was designed for each synthetic gene in the operon. Each gene cassette and regulatory region construct is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.

[0525] In certain embodiments the construct is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., FIG. 32). The insertion site may

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PCT/US2016/044922 be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. At the site of insertion, DNA primers that are homologous to the site of insertion and to the propionate construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting E. coli Nissle bacteria are genetically engineered to express a propionate biosynthesis cassette and produce propionate.

Example 4. Generation of Engineered Bacteria Comprising a transporter of

Propionate and/or a Propionate Catabolism Enzyme [0526] The pTet-prpE-PhaBCA plasmids (and other plasmids described herein) are transformed into E. coli Nissle, DH5a, or PIR1. All tubes, solutions, and cuvettes are prechilled to 4° C. An overnight culture of E. coli (Nissle, DH5a or PIR1) is diluted 1:100 in 4 mL of LB and grown until it reaches an OD600 of 0.4-0.6. ImL of the culture is then centrifuged at 13,000 rpm for 1 min in a 1.5mL microcentrifuge tube and the supernatant is removed. The cells are then washed three times in pre-chilled 10% glycerol and resuspended in 40uL pre-chilled 10% glycerol. The electroporator is set to 1.8kV. luL of a pTet-prpEPhaBCA miniprep is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled 1mm cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 500uL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing 50ug/mL Kanamycin for pTet-prpBCDE and pTet-mctC.

[0527] In alternate embodiments, the pTet-prpE-PhaBCA cassettes or pFNR-prpEPhaBCA cassettes may be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, MA).

[0528] To create a vector capable of integrating the synthesized the pTet-prpEPhaBCA or pFNR-prpE-PhaBCA cassettes into the chromosome, Gibson assembly is first used to add lOOObp sequences of DNA homologous to the a Nissle locues, e.g., the lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the locus, e.g., the lacZ locus in the Nissle genome. Gibson assembly is used to clone the fragment between these arms. PCR was used to amplify the region from

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PCT/US2016/044922 this plasmid containing the entire sequence of the homology arms, as well as the the prpEPhaBCA cassettes between them. This PCR fragment is used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells are grown out for 2 hours before plating on chloramphenicol at 20ug/mL at 37 degrees C. Growth at 37 degrees C also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus

Example 5. Lambda Red Recombination [0529] Lambda red recombination is used to make chromosomal modifications, e.g., to express one or more prpE-PhaBCA cassette(s) ( or other cassettes describd herein) in E. coli Nissle. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of E. coli. A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1:100 in 5 mL of LB media and grown until it reaches an OD600 of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to

2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.

[0530] DNA sequences comprising the desired prpE-PhaBCA cassette(s) shown above are ordered from a gene synthesis company. The lambda enzymes are used to insert this construct into the genome of E. coli Nissle through homologous recombination. The construct is inserted into a specific site in the genome of E. coli Nissle based on its DNA sequence. To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified, and includes approximately 50 bases on either side of the sequence. The homologous sequences are ordered as part of the synthesized gene. Alternatively, the homologous sequences may be added by PCR. The construct includes an

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PCT/US2016/044922 antibiotic resistance marker that may be removed by recombination. The resulting construct comprises approximately 50 bases of homology upstream, a kanamycin resistance marker that can be removed by recombination, the prpE-PhaBCA cassette(s), and approximately 50 bases of homology downstream.

Example 6. Establishment of Propionic Acidemia Biomarkers in the

PCCAA138T Hypomorph Mouse Model [0531] For in vivo studies, PCCAA138T hypomorph mice were obtained for use as a model for propionic acidemia. First, biomarkers for propionic acidemia were established.

[0532] PCCAA138T mice and FVB (parental) controls (10-12 weeks old) were kept on normal chow. Blood and urine were collected and were assayed for known biomarkers of propionic acidemia. In blood, the propionylcarnitine/acetylcamitine ratio, propionate concentration, and 2-methylcitrate concentration were determined by mass spectrometry as described herein. Results are shown in FIG. 6A-FIG. 6C. For urine, propionyl-glycine, Tigylglycine, and 2-methylcitrate were measured by LC-MS/MS as described herein, and results are shown in FIG. 6D-FIG. 6F.

Example 7. Enterorecirculation of Propionic Acid in the PCCAA138T

Hypomorph Mouse Model [0533] To determine whether propionate undergoes enterorecirculation, in a similar manner as has been hypothesized and shown for amino acids (see e.g., Chang et al., A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria; Artif Cells Blood Substit Immobil Biotechnol. 1995;23(1): 1-21), levels of enteroconversion of labeled propionate from the bloodstream were measured in various compartments of the gut using the PCCAA138T mouse model.

[0534] All PCCAA138T mice (10-12 weeks old) were kept on normal chow until O.lmg/g isotopic propionic acid was administered at TO by subcutaneous injection.

[0535] At each timepoint (0, 30 min, lh and 2h post-SC injection), animals [[(n=X)]] were euthanized, and blood, small intestine, large intesting and cecum, were removed and collected. Each intestinal section section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed

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PCT/US2016/044922 with 0.5 ml cold PBS and collected in a 1.5 ml tube. Blood was collected by by mandibular bleeding. Concentrations of endogenous and radiolabeled propionate in the blood, intestinal compartments, and cecum were measured by LC-MS/MS as described herein. As shown in FIG. 7A-FIG. 7D, isotopic propionic acid injected SC is seen at very low levels in the blood, small intestine, and cecum within 30 min, indicating that propionate has circulated from blood into the intestinal compartments in the the PA/MMA animal model.

Example 8. Bacterial Contribution to PA Biomarkers [0536] Experiments with antibiotic-treated PA patients suggest that bacterial metabolism in the gut contributes -30% of the propionate The bacterial contribution to levels of PA biomarkers are evaluated by measuring the effects of an antibiotic treatment which significantly reduces the microbiota population (>99.9%) in the PCCA model.

[0537] PCCAA138T mice are kept on normal chow until Day 1 of the study. On day 1, plasma, urine, fecal samples are taken and, antibiotics supplemented in water of half of the mice (Ampicilln (lg/L), Vancomycin (0.5 g/L), Neomycin (1 g/L), Metronidazole (1 g/L))

On D8, plasma, urine, fecal samples (n=4) are taken and metabolite levels quantified by LCMS/MS as described herein. Bacterial levels are quantified by qPCR using primers which amplify DNA from Nissle and total bacteria. Metabolites (propionate, propionlylcarnitine/acetylcarnitine ratio; propionylcarnitine, 2-methylcitrate, acetylcamitine, are quantified by LC-MS/MS as described herein.

Example 9. Polyhydroxyalkanoate (PHA) Pathway Propionate Consumption

Assay [0538] PHA pathway is a heterologous bacterial pathway used for carbon storage as polymers, and was assessed for its ability to consume propionate.

[0539] As described herein, the E. coli Nissle prpE gene and phaBCA genes from Acinetobacter sp RA3849 (codon optimized for expression in E. coli Nissle) were placed under the control of an aTc-inducible promoter in a single operon in a high copy plasmid, as shown in FIG. 10C and FIG. 11. Corresponding construct sequences are listed in Table 12 in Example 2. Next, the rate of propionate consumption of genetically engineered bacteria comprising the prpE-phaBCA circuit was assessed in vitro.

[0540] Cultures of E. coli Nissle transformed with the plasmid comprising the prpEphaBCA circuit driven by the tet promoter and cultures of wild type control Nissle were

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PCT/US2016/044922 grown overnight and then diluted 1:200 in LB. ATC was added to the cultures of the strain containing the prpE-phaBCA construct plasmid at a concentration of 100 ng/mL to induce expression of the prpE and phaBCA genes. Then, the cells were grown with shaking at 250 rpm. After 2 hrs of incubation, cells were pelleted down, washed, and resuspended in ImL M9 medium supplemented with glucose (0.2%) and propionate (2-8 mM) at a concentration of ~10⁹ cfu/ml bacteria. Aliquots were collected at 0 hrs, 1.5 hr, 3 hrs, and 4.5 hrs for propionate quantification as described herein. As shown in FIG. 12, the genetically engineered bacteria expressing prpE and phaBCA genes driven by the tet promoter are more efficient at removing propionate than wild type Nissle or the uninduced engineered strain.

The catabolic rate was calculated to be 0.396-1.4 umol hr’¹ per 10⁹ cells.

Example 10. PHA Pathway Performance with Mixed Organic Acids [0541] To determine whether acetate or butyrate (which are abundant in the colon) may have an effect on propionate consumption throught the PHA pathway, 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).

[0542] Cultures of E. coli Nissle transformed with the plasmid comprising the prpEphaBCA circuit driven by the tet promoter (as described in Example 9) and wild type control Nissle were grown overnight and then diluted 1:200 in LB. ATC was added to the cultures of the strain containing the prpE-phaBCA construct plasmid and the wild type Nissle cultures and cells were incubated for two hours. Cells were spun down and resuspended in as described in Example 9 in ImL M9 medium supplemented with glucose (0.2%) and propionate (6 mM), butyrate (4 MM), and acetate (10 mM) at a concentration of ~10⁹ cfu/ml bacteria. Aliquots were collected at 0 hrs, 1.5 hrs, 3 hrs, and 4.5 hrs for propionate quantification via LC-MS/MS as described herein. As shown in FIG. 13A, the genetically engineered bacteria expressing the tet-prpE and phaBCA gene cassette reduced the concentration of propionate compared to the wild type Nissle at a rate similar to the rate observed in the absence of acetate and butyrate in Example 9. The catabolic rate was calculated to be 0.396 -1.4 umol hr-1 per 109 cells.

[0543] Also, the genetically engineered bacteria did not affect acetate or butyrate levels as compared to wild type Nissle (FIG. 13B and FIG. 13C), indicating that the PHA pathway does not significantly affect acetate and butyrate concentrations.

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Example 11. Optimization of the PHA Pathway [0544] To optimize the PHA pathway and to determine the rate-limiting step in the pathway, the base strain expressing the aTc-inducible prpE-phaBCA operon was supplemented with a second plasmid expressing a construct containing one of the operon genes under the control of an arabinose inducible promoter, as shown in FIG. 14A-FIG.

14D. Table 14 lists the construct sequences from the additional plasmids.

[0545] In this assay, either the prpE-phaBCA operon alone, or both the prpE-phaBCA plasmid and the arabinose inducible plasmid carrying the additional copy of one of the genes in the pathway were induced to assess whether additional expression of any of the genes could increase propionate consumption. Wild type Nissle was included for reference.

Table 14. PHA Pathway Sequences - Additional Plasmid Constructs

Description	Sequence	SEQ ID NO
araC-Para-phaA (araC: lower case; RBS underlined; phaA: italics; L3S2P11 terminator: underlined bold; his terminator: bold)	ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatactc gcgagaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgatag gcatccgggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgcg ccagcttaatacgctaatccctaactgctggcggaacaaatgcgacagacgcgac ggcgacaggcagacatgctgtgcgacgctggcgatatcaaaattactgtctgcca ggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatgg agcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatc gccagcaattccgaatagcgcccttccccttgtccggcattaatgatttgcccaaac aggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaaccggtattgg caaatatcgacggccagttaagccattcatgccagtaggcgcgcggacgaaagta aacccactggtgataccattcgtgagcctccggatgacgaccgtagtgatgaatct ctccaggcgggaacagcaaaatatcacccggtcggcagacaaattctcgtccctg atttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattccc agcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacc cgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttgcgctt cagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcat CAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT TCTCGCTAACCCAACCGGTAACCCCGCTTATTAAA AGCATTCTGTAACAAAGCGGGACCAAAGCCATGA CAAAAACGCGTAACAAAAGTGTCTATAATCACGG CAGAAAAGTCCACATTGATTATTTGCACGGCGTCA CACTTTGCTATGCCATAGCATTTTTATCCATAAGA TTAGCGGATCCAGCCTGACGCTTTTTTTCGCAACT CTCTACTGTTTCTCCATACCATATTCATAGAAAGA ATACTAAGAGAGGTCAGAA7'GA/1/1GA7'G77'G77A7'C GTAGCCGCTAAACGCACTGCGATCGGTTCCTTTCTGG GGAGTCTGGCTTCCCTGAGCGCCCCTCAGTTGGGTC	SEQ ID NO: 40

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	AGACGGCTATCCGCGCAGTTTTGGATTCTGCAAATGT GAAACCAGAACAAGTGGACCAAGTAATTATGGGGAAT GTGCTGACCACCGGCGTTGGGCAAAATCCTGCTCGT CAGGCAGCAATCGCCGCTGGGATTCCTGTACAAGTT CCCGCCAGCACGCTTAATGTAGTGTGTGGGTCCGGA TTACGTGCCGTTCACCTGGCAGCTCAAGCCATCCAAT GCGATGAAGCCGATATCGTCGTTGCCGGAGGTCAAG AATCAATGTCCCAGTCTGCTCATTACATGCAGCTTCG CAATGGCCAGAAAATGGGTAACGCACAGTTAGTCGAT TCAATGGTGGCCGACGGCTTGACCGACGCGTATAAT CAATACCAGATGGGTATCACCGCGGAGAATATCGTCG AAAAACTTGGTCTTAATCGTGAAGAACAAGACCAGCT TGCTCTGACAAGTCAACAACGTGCTGCAGCAGCGCA GGCTGCCGGAAAATTCAAGGATGAAATTGCGGTCGTT TCGATTCCCCAGCGCAAAGGAGAGCCGGTCGTCTTC GCGGAAGACGAATATATCAAGGCCAATACCTCGTTGG AATCCTTGACGAAACTGCGTCCAGCATTCAAAAAAGA CGGTTCTGTTACAGCCGGCAACGCATCTGGCATTAAT GATGGGGCAGCCGCGGTCCTGATGATGTCCGCCGAC AAAGCGGCTGAACTGGGCTTAAAGCCTTTAGCACGCA TTAAAGGTTACGCGATGTCAGGAATTGAGCCGGAAAT CATGGGACTGGGTCCTGTAGACGCCGTTAAGAAAAC CCTTAATAAGGCTGGTTGGTCCTTAGACCAGGTCGAT CTGATCGAGGCCAATGAGGCTTTTGCTGCCCAAGCA CTGGGAGTAGCCAAGGAGCTTGGGCTGGACCTGGAC AAGGTAAATGTTAACGGAGGTGCGATCGCGCTGGGA CACCCGATCGGGGCTTCGGGTTGTCGTATCTTGGTC ACGTTATTACACGAAATGCAGCGTCGTGATGCAAAGA AGGGTATCGCCACATTGTGTGTGGGAGGTGGAATGG GGGTGGCGCTTGCCGTTGAGCGCGATTAAGGAGCV CGGTACCAAATTCCAGAAAAGAGACGCTTTCG
AGCGTCTTTTTTCGTTTTGGTCCGCGCAATAAA
AAAGCCCCCGGAAGGTGATCTTCCGGGGGCTT TCTCATGCGTT
araC-Para-phaB (araC: lower case; RBS underlined; phaB: italics; L3S2P11 terminator: underlined bold; his terminator: bold)	Ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatact cgcgagaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgata ggcatccgggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgc gccagcttaatacgctaatccctaactgctggcggaacaaatgcgacagacgcga cggcgacaggcagacatgctgtgcgacgctggcgatatcaaaattactgtctgcc aggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatg gagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttat cgccagcaattccgaatagcgcccttccccttgtccggcattaatgatttgcccaaa caggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaaccggtattg gcaaatatcgacggccagttaagccattcatgccagtaggcgcgcggacgaaagt aaacccactggtgataccattcgtgagcctccggatgacgaccgtagtgatgaatc tctccaggcgggaacagcaaaatatcacccggtcggcagacaaattctcgtccct gatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcc cagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaac	SEQ ID NO: 41

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	ccgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttgcgc ttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgca tCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT TCTCGCTAACCCAACCGGTAACCCCGCTTATTAAA AGCATTCTGTAACAAAGCGGGACCAAAGCCATGA CAAAAACGCGTAACAAAAGTGTCTATAATCACGG CAGAAAAGTCCACATTGATTATTTGCACGGCGTCA CACTTTGCTATGCCATAGCATTTTTATCCATAAGA TTAGCGGATCCAGCCTGACGCTTTTTTTCGCAACT CTCTACTGTTTCTCCATAccGCTAGAACTAGATCTA GAGT AAT AAGGAGGAAGGAATGTCAGAGCAGAAAG
TAGCTCTGGTTACCGGTGCGTTAGGTGGTATCGGAA GTGAGATCTGCCGCCAGCTTGTGACCGCCGGGTACA AGATTATCGCCACCGTTGTTCCACGCGAAGAAGACCG CGAAAAACAATGGTTGCAAAGTGAGGGGTTTCAAGAC TCTGATGTGCGTTTCGTATTAACAGATTTAAACAATCA CGAAGCTGCGACAGCGGCAATTCAAGAAGCGATTGC CGCCGAAGGACGCGTTGATGTATTGGTCAACAACGC GGGGATCACGCGCGATGCTACATTTAAGAAAATGTCC TATGAGCAATGGTCCCAAGTCATCGACACGAATTTAA AGACTCTTTTTACCGTGACCCAGCCAGTATTTAATAAA ATGCTTGAACAGAAGTCTGGCCGCATCGTAAACATTA GCTCTGTCAATGGTTTAAAAGGGCAATTTGGTCAAGC CAACTACTCGGCCTCGAAAGCAGGGATTATCGGGTTT ACTAAAGCATTGGCGCAGGAGGGTGCTCGCTCGAAC ATTTGCGTCAATGTCGTTGCTCCTGGTTACACAGCGA CACCCATGGTCACAGCAATGCGCGAGGATGTAATTAA GTCAATCGAAGCTCAAATTCCCCTGCAACGTCTGGCA GCACCGGCGGAGATTGCGGCAGCGGTTATGTATTTG GTGAGTGAACACGGTGCATACGTGACGGGCGAAACT TTGAGTATCAACGGCGGGCTGTACATGCACTAAGGA GCTCGGTACCAAATTCCAGAAAAGAGACGCTTT
CGAGCGTCTTTTTTCGTTTTGGTCCGCGCAATA
AAAAAGCCCCCGGAAGGTGATCTTCCGGGGGC TTTCTCATGCGTT
acaC-Para-phaC (araC: lower case; RBS underlined; phaC: italics; L3S2P11 terminator: underlined bold; his terminator: bold)	Ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatact cgcgagaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgata ggcatccgggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgc gccagcttaatacgctaatccctaactgctggcggaacaaatgcgacagacgcga cggcgacaggcagacatgctgtgcgacgctggcgatatcaaaattactgtctgcc aggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatg gagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttat cgccagcaattccgaatagcgcccttccccttgtccggcattaatgatttgcccaaa caggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaaccggtattg gcaaatatcgacggccagttaagccattcatgccagtaggcgcgcggacgaaagt aaacccactggtgataccattcgtgagcctccggatgacgaccgtagtgatgaatc tctccaggcgggaacagcaaaatatcacccggtcggcagacaaattctcgtccct gatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcc	SEQ ID NO: 42

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PCT/US2016/044922 cagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaac ccgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttgcgc ttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgca tCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT

TCTCGCTAACCCAACCGGTAACCCCGCTTATTAAA

AGCATTCTGTAACAAAGCGGGACCAAAGCCATGA

CAAAAACGCGTAACAAAAGTGTCTATAATCACGG

CAGAAAAGTCCACATTGATTATTTGCACGGCGTCA

CACTTTGCTATGCCATAGCATTTTTATCCATAAGA

TTAGCGGATCCAGCCTGACGCTTTTTTTCGCAACT

CTCTACTGTTTCTCCATACCACTATTATTTAATATA

CGACAACAGGAGGAACCAATGAATCCAAATTCCTTT

CGACGATATTTGGAAAAAACTGCAGGAATTTTACTATG

GACAATCGCCCATCAATGAAGCGTTGGCGCAGTTAAA

TAAGGAAGACATGAGTTTATTCTTCGAGGCGTTATCAA

AAAACCCTGCTCGTATGATGGAGATGCAGTGGTCCTG

GTGGCAAGGGCAGATTCAAATTTACCAGAACGTGTTA

ATGCGTAGTGTAGCCAAGGACGTAGCCCCCTTTATCC

AGCCAGAGTCCGGAGATCGTCGCTTCAACTCGCCAC

TTTGGC AAGAAC ATCC AAATTTTGATTT ACTGAGTCAA

TCCTACTTGTTGTTTTCTCAGTTGGTTCAAAATATGGT

GGATGTCGTTGAAGGAGTACCTGATAAGGTCCGCTAT

CGCATCCATTTCTTTACACGTCAGATGATCAATGCGTT

GTCTCCTTCTAATTTCCTGTGGACGAACCCTGAAGTA

ATTCAACAGACGGTCGCTGAACAGGGTGAGAATTTAG

TACGCGGGATGCAAGTATTTCACGATGATGTAATGAA

TTCGGGTAAATATTTGAGCATCCGTATGGTAAATAGC

GACAGTTTCTCTCTTGGCAAGGACTTGGCGTATACGC

CAGGAGCCGTAGTTTTCGAGAACGACATCTTTCAGCT

TCTTCAATACGAAGCCACAACCGAGAACGTATATCAA

ACCCCTATTCTTGTCGTACCTCCCTTCATCAACAAGTA

CTACGTGCTGGACCTGCGCGAACAGAATAGCTTGGTT

AATTGGCTGCGCCAACAAGGACATACGGTGTTTTTGA

TGTCGTGGCGTAACCCCAACGCAGAGCAGAAGGAGC

TTACCTTCGCTGACTTAATTACCCAAGGATCGGTAGA

AGCATTACGTGTTATCGAAGAAATCACGGGAGAGAAA

GAAGCTAACTGTATTGGATATTGCATCGGTGGTACAC

TTCTGGCTGCTACCCAGGCATATTATGTAGCTAAACG

CCTGAAAAATCACGTAAAGTCAGCGACTTATATGGCG

ACGATTATTGATTTTGAGAACCCCGGCTCATTGGGTG

TTTTCATTAATGAGCCGGTCGTAAGTGGACTTGAAAA

CCTTAATAATCAACTTGGTTACTTCGACGGGCGTCAA

CTTGCAGTGACATTTTCGTTGTTGCGCGAAAACACCT

TGTATTGGAATTATTACATCGATAATTACTTGAAGGGT

AAGGAACCGTCCGACTTTGACATCTTATACTGGAACT

CGGATGGTACGAATATCCCAGCAAAGATTCACAATTT

CCTGTTACGTAACCTTTATCTTAACAACGAACTTATTT

CTCCAAATGCCGTCAAAGTTAATGGTGTGGGTTTAAA

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	CCTTTCGCGCGTGAAGACTCCATCATTCTTCATTGCTA CGCAGGAGGACCATATCGCATTGTGGGATACCTGTTT TCGCGGCGCGGATTACCTGGGGGGTGAGAGCACACT TGTGCTTGGGGAAAGCGGACACGTCGCCGGCATTGT CAACCCGCCTTCTCGTAACAAGTATGGTTGTTACACG AACGCCGCCAAGTTTGAAAATACCAAGCAATGGCTTG ACGGTGCAGAATATCATCCCGAAAGCTGGTGGTTACG TTGGCAGGCATGGGTCACGCCTTATACTGGAGAGCA GGTTCCTGCGCGTAATTTGGGAAACGCACAGTACCC CAGTATTGAAGCGGCCCCTGGGCGTTATGTGCTGGT AAACCTG777TAAGGAGCTCGGTACCAAATTCCAG AAAAGAGACGCTTTCGAGCGTCTTTTTTCGTTT
TGGTCCGCGCAATAAAAAAGCCCCCGGAAGGT GATCTTCCGGGGGCTTTCTCATGCGTT
AraC-pAra-PrpE (AraC: Lower Case; RBS Underlined; PrpE: Italics; L3s2pll Terminator: Underlined; His Terminator: Bold)	ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatactc gcgagaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgatag gcatccgggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgcg ccagcttaatacgctaatccctaactgctggcggaacaaatgcgacagacgcgac ggcgacaggcagacatgctgtgcgacgctggcgatatcaaaattactgtctgcca ggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatgg agcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatc gccagcaattccgaatagcgcccttccccttgtccggcattaatgatttgcccaaac aggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaaccggtattgg caaatatcgacggccagttaagccattcatgccagtaggcgcgcggacgaaagta aacccactggtgataccattcgtgagcctccggatgacgaccgtagtgatgaatct ctccaggcgggaacagcaaaatatcacccggtcggcagacaaattctcgtccctg atttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattccc agcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacc cgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttgcgctt cagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcat CAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT TCTCGCTAACCCAACCGGTAACCCCGCTTATTAAA AGCATTCTGTAACAAAGCGGGACCAAAGCCATGA CAAAAACGCGTAACAAAAGTGTCTATAATCACGG CAGAAAAGTCCACATTGATTATTTGCACGGCGTCA CACTTTGCTATGCCATAGCATTTTTATCCATAAGA TTAGCGGATCCAGCCTGACGCTTTTTTTCGCAACT CTCTACTGTTTCTCCATACCAGATTTAAAGTAAGG CCAGGGAAAAAATGTCTTTTAGCGAATTTTATCAGCG TTCGATTAACGAACCGGAGAAGTTCTGGGCCGAGCA GGCCCGGCGTATTGACTGGCAGACGCCCTTTACGCA AACGCTCGACCACAGCAACCCGCCGTTTGCCCGTTG GTTTTGTGAAGGCCGAACCAACTTGTGTCACAACGCT ATCGACCGCTGGCTGGAGAAACAGCCAGAGGCGCTG GCATTGATTGCCGTCTCTTCGGAAACAGAGGAAGAGC GTACCTTTACCTTCCGCCAGTTACATGACGAAGTGAA TGCGGTGGCGTCAATGCTGCGCTCACTGGGCGTGCA GCGTGGCGATCGGGTGCTGGTGTATATGCCGATGAT	SEQ ID NO: 43

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TGCCGAAGCGCATATTACCCTGCTGGCCTGCGCGCG

CATTGGTGCTATTCACTCGGTGGTGTTTGGGGGATTT

GCTTCGCACAGCGTGGCAACGCGAATTGATGACGCT

AAACCGGTGCTGATTGTCTCGGCTGATGCCGGGGCG

CGCGGCGGTAAAATCATTCCGTATAAAAAATTGCTCG

ACGATGCGATAAGTCAGGCACAGCATCAGCCGCGTC

ACGTTTTACTGGTGGATCGCGGGCTGGCGAAAATGG

CGCGCGTTAGCGGGCGGGATGTCGATTTCGCGTCGT

TGCGCCATCAACACATCGGCGCGCGGGTGCCGGTG

GCATGGCTGGAATCCAACGAAACCTCCTGCATTCTCT

ACACCTCCGGCACGACCGGCAAACCTAAAGGTGTGC

AGCGTGATGTCGGCGGATATGCGGTGGCGCTGGCG

GGCGTGTTCTTTTGTGCTTCGGATATCGGCTGGGTGG

TAGGGCATTCGTATATCGTTTACGCGCCGCTGCTGGC

GGGGATGGCGACTATCGTTTACGAAGGATTGCCGAC

CTGGCCGGACTGCGGCGTGTGGTGGAAAATTGTCGA

GAAATATCAGGTTAGCCGCATGTTCTCAGCGCCGACC

GCCATTCGCGTGCTGAAAAAATTCCCTACCGCTGAAA

TTCGCAAACACGATCTTTCGTCGCTGGAAGTGCTCTA

TCTGGCTGGAGAACCGCTGGACGAGCCGACCGCCA

GTTGGGTGAGCAATACGCTGGATGTGCCGGTCATCG

ACAACTACTGGCAGACCGAATCCGGCTGGCCGATTAT

GGCGATTGCTCGCGGTCTGGATGACAGACCGACGCG

TCTGGGAAGCCCCGGCGTGCCGATGTATGGCTATAA

CGTGCAGTTGCTCAATGAAGTCACCGGCGAACCGTG

TGGCGTCAATGAGAAAGGGATGCTGGTAGTGGAGGG

GCCATTGCCGCCAGGCTGTATTCAAACCATCTGGGG

CGACGACGACCGCTTTGTGAAGACGTACTGGTCGCT

GTTTTCCCGTCCGGTGTACGCCACTTTTGACTGGGGC

ATCCGCGATGCTGACGGTTATCACTTTATTCTCGGGC

GCACTGACGATGTGATTAACGTTGCCGGACATCGGCT

GGGTACGCGTGAGATTGAAGAGAGTATCTCCAGTCAT

CCGGGCGTTGCCGAAGTGGCGGTGGTTGGGGTGAA

AGATGCGCTGAAAGGGCAGGTGGCGGTGGCGTTTGT

CATTCCGAAAGAGAGCGACAGTCTGGAAGACCGTGA

GGTGGCGCACTCGCAAGAGAAGGCGATTATGGCGCT

GGTGGACAGCCAGATTGGCAACTTTGGCCGCCCGGC

GCACGTCTGGTTTGTCTCGCAATTGCCAAAAACGCGA

TCCGGAAAAATGCTGCGCCGCACGATCCAGGCGATT

TGCGAAGGACGCGATCCTGGGGATCTGACGACCATT

GATGATCCGGCGTCGTTGGATCAGATCCGCCAGGCG

ATGGAAGAG7AGGGAGCTCGGTACCAAATTCCAG

AAAAGAGACGCTTTCGAGCGTCTTTTTTCGTTT

TGGTCCGCGCAATAAAAAAGCCCCCGGAAGGT

GATCTTCCGGGGGCTTTCTCATGCGTT

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PCT/US2016/044922 [0546] Cultures of E. coli Nissle transformed with the plasmid comprising the tetprpE-phaBCA circuit and the second plasmid (containing one of pAra-prpE or pAra-phaB or pAra-phaC or pAra-phaA) were grown overnight and then diluted 1:200 in LB. Wild type control Nissle cultures were also grown as a referene. ATC (100 ng/mL) was added to induce the tet- prpE-phaBCA construct gene cassette. In half of the cultures of the four strains containing the tet-prpE-phaBCA circuit, arabinose was added at a concentration of 10 mM to induce the second plasmid. Cells were grown with shaking at 250 rpm. After 2 hrs of incubation, cells were pelleted down, washed, and resuspended in ImL M9 medium 0.5% glucose 8 mM propionate added at a concentration of ~10⁹ cfu/ml bacteria. Aliquots were collected at 0 hrs, lhrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs for propionate quantification by LCMS/MS. As shown in FIG. 14A-FIG. 14D, the rate of propionate consumption is increased most significantly when more phaC is expressed, suggesting that the pathway is improved by increasing the PhaC levels from the original prpE-phaBCA plasmid.

[0547] In certain embodiments, the prpE-phaBCA circuit is futher modified by adding a strong RBS upstream of the phaC translation start site. In certain embodiments, the genetically engineered bacteria comprise one or more prpE-phaBCA gene cassettes and one or more additional cassettes comprising the phaA gene.

Example 12. In vitro Activity of the MMCA Pathway [0548] The methylmalonyl-CoA pathway was assessed in vitro for its ability to catabolize propionate. As described in Example 3, genes accA (from Streptomyces coelicolor), pccB (from Streptomyces coelicolor), mmcE (from Propionibacterium freudenreichii). and mutAB (from Propionibacterium freudenreichii) were codon-optimized for expression in E. coli Nissle. Two plasmids, the first plasmid with a cassette comprising prpE, pccB, accAl, under the control of an inducible Ptet promoter and the second plasmid with a cassette comprising mmcE and mutAB under the control of a second inducible promoter, Para, were generated (as shown in FIG 15C and FIG 16A and FIG. 16B). Induction of the pathway therefore requires the addition of aTc and arabinose. Sequences of MMCA pathway circuits are listed in Table 13 in Example 3.

[0549] Cultures of E. coli Nissle comprising the first and second plasmids with the MMCA circuits and wild type control Nissle, were grown overnight in LB and 50ug/mL Ampicillin and then diluted 1:100 in LB. The cells were grown with shaking (250 rpm) to early log phase with the appropriate antibiotics. Anhydrous tetracycline (ATC) and arabinose

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PCT/US2016/044922 (10 mM) was added to cultures at a concentration of 100 ng/mL to induce expression of the constructs, and bacteria were grown for another 2 hours. Bacteria were then pelleted, washed, and resuspended in minimal media at ~10⁹ cfu/ml, and supplemented with 0.5% glucose and propionate (6 mM). Aliquots were removed at 0 hrs, 2 hrs, 4, hrs, 17, hrs and 18 hrs for propionate quantification by LC-MS/MS analysis.

[0550] For induction of the PHA pathway, cultures were grown, induced, and assayed as described in Example 9.

[0551] As shown in FIG. 18, the expression of the MMCA circuits reduces the propionate concentration in the media, indicating that the cicuits promote propionate catalysis. Propionate assay was initiated with ~10⁹ cfu/ml pre-induced bacteria and the propionate consumption rate was -3.8 μΜ/hr/lO⁹ bacteria in the strain expressing the methylmalonyl-CoA pathway circuit. Overall the MMCA pathway seems more effective at propionate breakdown than the PHA pathway.

Example 13. In vitro Activity of the MMCA pathway Circuit in Combination with a Succinate Exporter Circuit [0552] In order to determine whether a succinate exporter may increase the amount of propionate catabolized through the MMCA pathway, a construct was generated comprising the sucEl succinate exporter (from Corynebacterium glutamicum (as shown in FIG. 17B and FIG. 17D) or the E. coli dcuC succinate transporter (FIG. 17E) or comprising both transporters (FIG. 17F). The sucEl construct was placed under the control of Para (arabinose-inducible) in the Nissle chromosome. This knock-in also deleted the araBA genes as well as part of the araD gene, effectively eliminating metabolism of arabinose by E. coli.

[0553] Sequences of the exporter constructs are shown in Table 15. In vitro activity of MMCA pathway circuit is compared alone or in combination with an integrated sucEl circuit, essentially as described in Example 12 and elsewhere herein.

Table 15. Succinate Exporter Construct Sequences

Description	Sequence	SEQ ID NO
pAraC-SucEl (as shown in FIG. 17D; AraC: lower case; pARA: upper case	Ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatactcgc gagaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgataggcatcc gggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgcgccagcttaata cgctaatccctaactgctggcggaacaaatgcgacagacgcgacggcgacaggcaga catgctgtgcgacgctggcgatatcaaaattactgtctgccaggtgatcgctgatgtactg acaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccat	SEQ ID NO: 44

-239WO 2017/023818

PCT/US2016/044922 italics; RBS: underlined; sucEl: bold; FRT minimal: underline italics) gcgccgcagtaacaattgctcaagcagatttatcgccagcaattccgaatagcgcccttc cccttgtccggcattaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttca tccgggcgaaagaaaccggtattggcaaatatcgacggccagttaagccattcatgcca gtaggcgcgcggacgaaagtaaacccactggtgataccattcgtgagcctccggatga cgaccgtagtgatgaatctctccaggcgggaacagcaaaatatcacccggtcggcaga caaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataa cctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggc gttaaacccgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttg cgcttcagccaiACTTTTCATACTCCCGCCATTCAGAGAAGAAA

CCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGT

CTTTTACTGGCTCTTCTCGCTAACCCAACCGGTAACCCC

GCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGC

CATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC

AGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTT

GCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCC

7ACCCGTTTTTTTGGATGGAGTGAAACGATGTCCTTC

CTGGTCGAGAATCAATTGTTAGCACTTGTCGTGAT

CATGACCGTCGGGCTTTTACTTGGACGTATCAAAA

TCTTTGGTTTCCGTTTGGGTGTGGCCGCCGTGTTG

TTCGTCGGCCTTGCTTTAAGCACCATTGAGCCCGA

CATTTCGGTTCCATCCCTTATTTACGTGGTTGGCC

TTTCGCTTTTTGTGTATACTATCGGTCTGGAAGCT

GGCCCCGGTTTTTTTACATCTATGAAGACGACGGG

TTTGCGCAATAACGCACTGACGTTAGGTGCCATTA

TCGCGACAACAGCACTTGCGTGGGCACTGATTAC

CGTCTTGAATATTGATGCCGCCTCAGGAGCTGGTA

TGCTTACTGGTGCCTTAACTAATACGCCCGCTATG

GCTGCGGTAGTGGATGCACTTCCCTCATTAATTGA

TGACACAGGCCAGCTGCATCTTATTGCTGAGCTGC

CGGTGGTTGCTTATTCCCTGGCTTATCCCTTGGGG

GTACTGATTGTGATCTTGAGCATCGCCATCTTTTC

TTCAGTGTTTAAGGTTGACCATAACAAGGAGGCAG

AAGAGGCTGGGGTAGCGGTCCAAGAACTTAAGGG

CCGCCGTATCCGCGTAACTGTAGCTGACTTGCCAG

CCCTTGAGAACATTCCTGAGTTGCTTAATTTACAT

GTTATCGTCTCGCGTGTAGAGCGCGACGGAGAGC

AGTTCATCCCCTTATATGGCGAACATGCACGCATC

GGCGATGTACTGACTGTCGTGGGGGCCGACGAGG

AACTGAACCGCGCGGAAAAAGCCATCGGAGAGTT

AATTGACGGTGATCCTTACTCTAACGTTGAACTGG

ACTATCGTCGTATCTTCGTCTCTAATACGGCGGTT

GTCGGTACACCCCTGAGCAAATTGCAACCGCTTTT

TAAAGATATGCTTATTACTCGCATTCGCCGCGGTG

ATACGGATCTGGTAGCTTCCTCGGACATGACGCTT

CAATTAGGCGACCGCGTTCGTGTGGTTGCCCCAG

CCGAGAAACTTCGTGAAGCGACTCAGTTGCTTGG

AGACTCTTACAAAAAGCTGTCCGACTTTAATTTAT

TGCCTCTTGCTGCGGGCTTAATGATTGGCGTCCTT

-240WO 2017/023818

PCT/US2016/044922

	GTTGGAATGGTTGAATTCCCACTGCCTGGGGGGT CATCTTTAAAACTTGGCAATGCCGGTGGTCCGTTG GTTGTCGCGCTGTTGCTTGGGATGATCAATCGTAC GGGAAAGTTCGTCTGGCAGATCCCGTACGGAGCA AACTTGGCGTTACGTCAGTTGGGTATCACCCTGTT CTTGGCGGCTATTGGCACTTCCGCGGGAGCTGGG TTTCGCTCAGCTATTAGCGACCCGCAATCTCTGAC CATTATTGGATTTGGTGCGTTGTTAACCTTGTTTA TTAGTATTACCGTCTTGTTCGTTGGGCATAAGTTG ATGAAAATCCCGTTTGGGGAAACGGCGGGTATCT TAGCTGGAACGCAGACCCATCCAGCAGTATTATCA TATGTGTCTGACGCATCTCGCAACGAGTTGCCAGC CATGGGGTACACCTCAGTGTATCCCTTGGCTATGA TTGCGAAAATCCTGGCTGCACAAACACTTTTGTTT CTGTTGATTtaatgaGGAATCGACTCCACGTCCCTAGCG TGTGTAGGCTGGAGCTGCTTCGAAGTTCCrArACTTTCr AGAGAATAGGAACTTC
SucEl with RBS (underlined)	CCCGTTTTTTTGGATGGAGTGAAACGATGTCCTTCCT	SEQ ID NO: 45
GGTCGAGAATCAATTGTTAGCACTTGTCGTGATCATG ACCGTCGGGCTTTTACTTGGACGTATCAAAATCTTTG GTTTCCGTTTGGGTGTGGCCGCCGTGTTGTTCGTCGG CCTTGCTTTAAGCACCATTGAGCCCGACATTTCGGTT CCATCCCTTATTTACGTGGTTGGCCTTTCGCTTTTTGT GTATACTATCGGTCTGGAAGCTGGCCCCGGTTTTTTT ACATCTATGAAGACGACGGGTTTGCGCAATAACGCA CTGACGTTAGGTGCCATTATCGCGACAACAGCACTTG CGTGGGCACTGATTACCGTCTTGAATATTGATGCCGC CTCAGGAGCTGGTATGCTTACTGGTGCCTTAACTAAT ACGCCCGCTATGGCTGCGGTAGTGGATGCACTTCCCT CATTAATTGATGACACAGGCCAGCTGCATCTTATTGC TGAGCTGCCGGTGGTTGCTTATTCCCTGGCTTATCCCT TGGGGGTACTGATTGTGATCTTGAGCATCGCCATCTT TTCTTCAGTGTTTAAGGTTGACCATAACAAGGAGGCA GAAGAGGCTGGGGTAGCGGTCCAAGAACTTAAGGGC CGCCGTATCCGCGTAACTGTAGCTGACTTGCCAGCCC TTGAGAACATTCCTGAGTTGCTTAATTTACATGTTATC GTCTCGCGTGTAGAGCGCGACGGAGAGCAGTTCATC CCCTTATATGGCGAACATGCACGCATCGGCGATGTAC TGACTGTCGTGGGGGCCGACGAGGAACTGAACCGCG CGGAAAAAGCCATCGGAGAGTTAATTGACGGTGATC CTTACTCTAACGTTGAACTGGACTATCGTCGTATCTTC GTCTCTAATACGGCGGTTGTCGGTACACCCCTGAGCA AATTGCAACCGCTTTTTAAAGATATGCTTATTACTCG CATTCGCCGCGGTGATACGGATCTGGTAGCTTCCTCG GACATGACGCTTCAATTAGGCGACCGCGTTCGTGTGG TTGCCCCAGCCGAGAAACTTCGTGAAGCGACTCAGTT GCTTGGAGACTCTTACAAAAAGCTGTCCGACTTTAAT TTATTGCCTCTTGCTGCGGGCTTAATGATTGGCGTCCT

-241WO 2017/023818

PCT/US2016/044922

	TGTTGGAATGGTTGAATTCCCACTGCCTGGGGGGTCA TCTTTAAAACTTGGCAATGCCGGTGGTCCGTTGGTTG TCGCGCTGTTGCTTGGGATGATCAATCGTACGGGAAA GTTCGTCTGGCAGATCCCGTACGGAGCAAACTTGGCG TTACGTCAGTTGGGTATCACCCTGTTCTTGGCGGCTA TTGGCACTTCCGCGGGAGCTGGGTTTCGCTCAGCTAT TAGCGACCCGCAATCTCTGACCATTATTGGATTTGGT GCGTTGTTAACCTTGTTTATTAGTATTACCGTCTTGTT CGTTGGGCATAAGTTGATGAAAATCCCGTTTGGGGAA ACGGCGGGTATCTTAGCTGGAACGCAGACCCATCCA GCAGTATTATCATATGTGTCTGACGCATCTCGCAACG AGTTGCCAGCCATGGGGTACACCTCAGTGTATCCCTT GGCTATGATTGCGAAAATCCTGGCTGCACAAACACTT TTGTTTCTGTTGATT
SucEl	ATGTCCTTCCTGGTCGAGAATCAATTGTTAGCACTTG TCGTGATCATGACCGTCGGGCTTTTACTTGGACGTAT CAAAATCTTTGGTTTCCGTTTGGGTGTGGCCGCCGTG TTGTTCGTCGGCCTTGCTTTAAGCACCATTGAGCCCG ACATTTCGGTTCCATCCCTTATTTACGTGGTTGGCCTT TCGCTTTTTGTGTATACTATCGGTCTGGAAGCTGGCC CCGGTTTTTTTACATCTATGAAGACGACGGGTTTGCG CAATAACGCACTGACGTTAGGTGCCATTATCGCGACA ACAGCACTTGCGTGGGCACTGATTACCGTCTTGAATA TTGATGCCGCCTCAGGAGCTGGTATGCTTACTGGTGC CTTAACTAATACGCCCGCTATGGCTGCGGTAGTGGAT GCACTTCCCTCATTAATTGATGACACAGGCCAGCTGC ATCTTATTGCTGAGCTGCCGGTGGTTGCTTATTCCCTG GCTTATCCCTTGGGGGTACTGATTGTGATCTTGAGCA TCGCCATCTTTTCTTCAGTGTTTAAGGTTGACCATAAC AAGGAGGCAGAAGAGGCTGGGGTAGCGGTCCAAGA ACTTAAGGGCCGCCGTATCCGCGTAACTGTAGCTGAC TTGCCAGCCCTTGAGAACATTCCTGAGTTGCTTAATT TACATGTTATCGTCTCGCGTGTAGAGCGCGACGGAGA GCAGTTCATCCCCTTATATGGCGAACATGCACGCATC GGCGATGTACTGACTGTCGTGGGGGCCGACGAGGAA CTGAACCGCGCGGAAAAAGCCATCGGAGAGTTAATT GACGGTGATCCTTACTCTAACGTTGAACTGGACTATC GTCGTATCTTCGTCTCTAATACGGCGGTTGTCGGTAC ACCCCTGAGCAAATTGCAACCGCTTTTTAAAGATATG CTTATTACTCGCATTCGCCGCGGTGATACGGATCTGG TAGCTTCCTCGGACATGACGCTTCAATTAGGCGACCG CGTTCGTGTGGTTGCCCCAGCCGAGAAACTTCGTGAA GCGACTCAGTTGCTTGGAGACTCTTACAAAAAGCTGT CCGACTTTAATTTATTGCCTCTTGCTGCGGGCTTAATG ATTGGCGTCCTTGTTGGAATGGTTGAATTCCCACTGC CTGGGGGGTCATCTTTAAAACTTGGCAATGCCGGTGG TCCGTTGGTTGTCGCGCTGTTGCTTGGGATGATCAAT CGTACGGGAAAGTTCGTCTGGCAGATCCCGTACGGA	SEQ ID NO: 46

-242WO 2017/023818

PCT/US2016/044922

	GCAAACTTGGCGTTACGTCAGTTGGGTATCACCCTGT TCTTGGCGGCTATTGGCACTTCCGCGGGAGCTGGGTT TCGCTCAGCTATTAGCGACCCGCAATCTCTGACCATT ATTGGATTTGGTGCGTTGTTAACCTTGTTTATTAGTAT TACCGTCTTGTTCGTTGGGCATAAGTTGATGAAAATC CCGTTTGGGGAAACGGCGGGTATCTTAGCTGGAACG CAGACCCATCCAGCAGTATTATCATATGTGTCTGACG CATCTCGCAACGAGTTGCCAGCCATGGGGTACACCTC AGTGTATCCCTTGGCTATGATTGCGAAAATCCTGGCT GCACAAACACTTTTGTTTCTGTTGATT
pAraC-dcuC (as shown in FIG. 17E; AraC: lower case; pARA: upper case italics; RBS: underlined; dcuC: bold; FRT minimal: underline italics)	Ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatactcgc gagaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgataggcatcc gggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgcgccagcttaata cgctaatccctaactgctggcggaacaaatgcgacagacgcgacggcgacaggcaga catgctgtgcgacgctggcgatatcaaaattactgtctgccaggtgatcgctgatgtactg acaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccat gcgccgcagtaacaattgctcaagcagatttatcgccagcaattccgaatagcgcccttc cccttgtccggcattaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttca tccgggcgaaagaaaccggtattggcaaatatcgacggccagttaagccattcatgcca gtaggcgcgcggacgaaagtaaacccactggtgataccattcgtgagcctccggatga cgaccgtagtgatgaatctctccaggcgggaacagcaaaatatcacccggtcggcaga caaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataa cctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggc gttaaacccgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttg cgcttcagccatACTTTTCATACTCCCGCCATTCAGAGAAGAAA CCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGT CTTTTACTGGCTCTTCTCGCTAACCCAACCGGTAACCCC GCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGC CATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC AGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTT GCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCC AGCCTGACGCTTTTTTTCGCAACTCTCTACTGTTTCTCCA 7ACCCGGGGCCCAATAGGCTCCCTATAAGAGATAGAA	SEQ ID NO: 47
CTATGCTGACATTCATTGAACTCCTTATTGGGGTT GTGGTTATTGTGGGTGTAGCTCGCTACATCATTAA AGGGTATTCTGCCACTGGCGTGTTATTTGTCGGTG GCCTGTTATTGCTGATTATCAGTGCCATTATGGGG CACAAAGTGTTACCGTCCAGCCAGGCTTCAACAG GCTACAGCGCCACGGATATCGTTGAATACGTTAAA ATATTGCTAATGAGCCGCGGCGGCGACCTCGGCA TGATGATTATGATGCTGTGTGGCTTTGCCGCTTAC ATGACCCATATCGGCGCGAATGATATGGTGGTCA AGCTGGCGTCAAAACCATTGCAGTATATTAACTCC CCTTACCTTCTGATGATTGCCGCCTATTTTGTTGC CTGTCTGATGTCACTGGCCGTCTCTTCCGCAACCG GTCTGGGTGTTTTGCTGATGGCAACCCTGTTTCCG GTGATGGTAAACGTTGGTATCAGTCGTGGCGCAG CTGCTGCCATTTGTGCCTCCCCGGCGGCGATTATT

-243WO 2017/023818

PCT/US2016/044922

	CTCGCACCGACTTCAGGGGATGTGGTGCTGGCGG CGCAGGCTTCCGAAATGTCGCTGATTGACTTCGCC TTCAAAACAACGCTGCCTATCTCAATTGCTGCAAT TATCGGCATGGCGATCGCCCACTTCTTCTGGCAAC GTTATCTGGATAAAAAAGAGCACATCTCTCATGAA ATGTTAGATGTCAGTGAAATCACCACCACTGCCCC TGCGTTTTATGCCATTTTGCCGTTCACGCCGATCA TCGGAGTACTGATTTTTGACGGCAAATGGGGTCC GCAATTACACATCATCACTATTCTGGTGATTTGTA TGCTAATTGCCTCCATTCTGGAGTTCATCCGCAGC TTTAATACCCAGAAAGTTTTCTCTGGTCTGGAAGT GGCTTATCGCGGTATGGCAGATGCATTTGCTAACG TGGTGATGCTGCTGGTTGCCGCTGGGGTATTCGC TCAGGGGCTTAGCACCATCGGCTTTATTCAAAGTC TGATTTCTATCGCTACCTCGTTTGGTTCGGCGAGT ATCATCCTGATGCTGGTATTGGTGATCCTGACAAT GCTGGCGGCAGTCACGACCGGTTCAGGCAATGCG CCGTTTTATGCGTTTGTTGAGATGATCCCGAAACT GGCGCACTCCTCCGGCATTAACCCGGCGTATTTGA CTATCCCGATGCTGCAGGCGTCAAACCTGGGTCG TACCCTATCACCCGTTTCTGGCGTAGTCGTTGCGG TTGCCGGGATGGCGAAGATCTCGCCGTTTGAAGT CGTAAAACGCACCTCGGTGCCGGTGCTTGTTGGTT TGGTGATTGTTATCGTTGCTACAGAGCTGATGGTG CCAGGAACGGCAGCAGCGGTCACAGGCAAGTAAG GAATCGACTCCACGTCCCTAGCGTGTGTAGGCTGGAG CAGCAACGAAGTTCCTATACTTTCTAGAGAATAGGAACTT C
dcuC with RBS (underlined)	GGGCCCAATAGGCTCCCTATAAGAGATAGAACTATG	SEQ ID NO: 48
CTGACATTCATTGAACTCCTTATTGGGGTTGTGGTTAT TGTGGGTGTAGCTCGCTACATCATTAAAGGGTATTCT GCCACTGGCGTGTTATTTGTCGGTGGCCTGTTATTGCT GATTATCAGTGCCATTATGGGGCACAAAGTGTTACCG TCCAGCCAGGCTTCAACAGGCTACAGCGCCACGGAT ATCGTTGAATACGTTAAAATATTGCTAATGAGCCGCG GCGGCGACCTCGGCATGATGATTATGATGCTGTGTGG CTTTGCCGCTTACATGACCCATATCGGCGCGAATGAT ATGGTGGTCAAGCTGGCGTCAAAACCATTGCAGTATA TTAACTCCCCTTACCTTCTGATGATTGCCGCCTATTTT GTTGCCTGTCTGATGTCACTGGCCGTCTCTTCCGCAA CCGGTCTGGGTGTTTTGCTGATGGCAACCCTGTTTCC GGTGATGGTAAACGTTGGTATCAGTCGTGGCGCAGCT GCTGCCATTTGTGCCTCCCCGGCGGCGATTATTCTCG CACCGACTTCAGGGGATGTGGTGCTGGCGGCGCAGG CTTCCGAAATGTCGCTGATTGACTTCGCCTTCAAAAC AACGCTGCCTATCTCAATTGCTGCAATTATCGGCATG GCGATCGCCCACTTCTTCTGGCAACGTTATCTGGATA AAAAAGAGCACATCTCTCATGAAATGTTAGATGTCA

-244WO 2017/023818

PCT/US2016/044922

	GTGAAATCACCACCACTGCCCCTGCGTTTTATGCCAT TTTGCCGTTCACGCCGATCATCGGAGTACTGATTTTT GACGGCAAATGGGGTCCGCAATTACACATCATCACT ATTCTGGTGATTTGTATGCTAATTGCCTCCATTCTGGA GTTCATCCGCAGCTTTAATACCCAGAAAGTTTTCTCT GGTCTGGAAGTGGCTTATCGCGGTATGGCAGATGCAT TTGCTAACGTGGTGATGCTGCTGGTTGCCGCTGGGGT ATTCGCTCAGGGGCTTAGCACCATCGGCTTTATTCAA AGTCTGATTTCTATCGCTACCTCGTTTGGTTCGGCGA GTATCATCCTGATGCTGGTATTGGTGATCCTGACAAT GCTGGCGGCAGTCACGACCGGTTCAGGCAATGCGCC GTTTTATGCGTTTGTTGAGATGATCCCGAAACTGGCG CACTCCTCCGGCATTAACCCGGCGTATTTGACTATCC CGATGCTGCAGGCGTCAAACCTGGGTCGTACCCTATC ACCCGTTTCTGGCGTAGTCGTTGCGGTTGCCGGGATG GCGAAGATCTCGCCGTTTGAAGTCGTAAAACGCACCT CGGTGCCGGTGCTTGTTGGTTTGGTGATTGTTATCGTT GCTACAGAGCTGATGGTGCCAGGAACGGCAGCAGCG GTCACAGGCAAGTAA
dcuC	ATGCTGACATTCATTGAACTCCTTATTGGGGTTGTGG TTATTGTGGGTGTAGCTCGCTACATCATTAAAGGGTA TTCTGCCACTGGCGTGTTATTTGTCGGTGGCCTGTTAT TGCTGATTATCAGTGCCATTATGGGGCACAAAGTGTT ACCGTCCAGCCAGGCTTCAACAGGCTACAGCGCCAC GGATATCGTTGAATACGTTAAAATATTGCTAATGAGC CGCGGCGGCGACCTCGGCATGATGATTATGATGCTGT GTGGCTTTGCCGCTTACATGACCCATATCGGCGCGAA TGATATGGTGGTCAAGCTGGCGTCAAAACCATTGCAG TATATTAACTCCCCTTACCTTCTGATGATTGCCGCCTA TTTTGTTGCCTGTCTGATGTCACTGGCCGTCTCTTCCG CAACCGGTCTGGGTGTTTTGCTGATGGCAACCCTGTT TCCGGTGATGGTAAACGTTGGTATCAGTCGTGGCGCA GCTGCTGCCATTTGTGCCTCCCCGGCGGCGATTATTC TCGCACCGACTTCAGGGGATGTGGTGCTGGCGGCGC AGGCTTCCGAAATGTCGCTGATTGACTTCGCCTTCAA AACAACGCTGCCTATCTCAATTGCTGCAATTATCGGC ATGGCGATCGCCCACTTCTTCTGGCAACGTTATCTGG ATAAAAAAGAGCACATCTCTCATGAAATGTTAGATGT CAGTGAAATCACCACCACTGCCCCTGCGTTTTATGCC ATTTTGCCGTTCACGCCGATCATCGGAGTACTGATTT TTGACGGCAAATGGGGTCCGCAATTACACATCATCAC TATTCTGGTGATTTGTATGCTAATTGCCTCCATTCTGG AGTTCATCCGCAGCTTTAATACCCAGAAAGTTTTCTC TGGTCTGGAAGTGGCTTATCGCGGTATGGCAGATGCA TTTGCTAACGTGGTGATGCTGCTGGTTGCCGCTGGGG TATTCGCTCAGGGGCTTAGCACCATCGGCTTTATTCA AAGTCTGATTTCTATCGCTACCTCGTTTGGTTCGGCG AGTATCATCCTGATGCTGGTATTGGTGATCCTGACAA	SEQ ID NO: 49

-245WO 2017/023818

PCT/US2016/044922

	TGCTGGCGGCAGTCACGACCGGTTCAGGCAATGCGC CGTTTTATGCGTTTGTTGAGATGATCCCGAAACTGGC GCACTCCTCCGGCATTAACCCGGCGTATTTGACTATC CCGATGCTGCAGGCGTCAAACCTGGGTCGTACCCTAT CACCCGTTTCTGGCGTAGTCGTTGCGGTTGCCGGGAT GGCGAAGATCTCGCCGTTTGAAGTCGTAAAACGCAC CTCGGTGCCGGTGCTTGTTGGTTTGGTGATTGTTATCG TTGCTACAGAGCTGATGGTGCCAGGAACGGCAGCAG CGGTCACAGGCAAGTAA
Para-sucE-dcuC construct (as shown in FIG. 17F; AraC: lower case; pARA: upper case italics; RBS: underlined; sucE: bold; dcuC: bold underlined; FRT minimal: underline italics)	ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatactcgcg agaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgataggcatccg ggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgcgccagcttaatac gctaatccctaactgctggcggaacaaatgcgacagacgcgacggcgacaggcagac atgctgtgcgacgctggcgatatcaaaattactgtctgccaggtgatcgctgatgtactga caagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatg cgccgcagtaacaattgctcaagcagatttatcgccagcaattccgaatagcgcccttcc ccttgtccggcattaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcat ccgggcgaaagaaaccggtattggcaaatatcgacggccagttaagccattcatgcca gtaggcgcgcggacgaaagtaaacccactggtgataccattcgtgagcctccggatga cgaccgtagtgatgaatctctccaggcgggaacagcaaaatatcacccggtcggcaga caaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataa cctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggc gttaaacccgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttg cgcttcagccatACTTTTCATACTCCCGCCATTCAGAGAAGAAA CCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGT CTTTTACTGGCTCTTCTCGCTAACCCAACCGGTAACCCC GCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGC CATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC AGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTT GCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCC AGCCTGACGCTTTTTTTCGCAACTCTCTACTGTTTCTCCA 7ACCCGTTTTTTTGGATGGAGTGAAACGATGTCCTTC CTGGTCGAGAATCAATTGTTAGCACTTGTCGTGAT CATGACCGTCGGGCTTTTACTTGGACGTATCAAAA TCTTTGGTTTCCGTTTGGGTGTGGCCGCCGTGTTG TTCGTCGGCCTTGCTTTAAGCACCATTGAGCCCGA CATTTCGGTTCCATCCCTTATTTACGTGGTTGGCC TTTCGCTTTTTGTGTATACTATCGGTCTGGAAGCT GGCCCCGGTTTTTTTACATCTATGAAGACGACGGG TTTGCGCAATAACGCACTGACGTTAGGTGCCATTA TCGCGACAACAGCACTTGCGTGGGCACTGATTAC CGTCTTGAATATTGATGCCGCCTCAGGAGCTGGTA TGCTTACTGGTGCCTTAACTAATACGCCCGCTATG GCTGCGGTAGTGGATGCACTTCCCTCATTAATTGA TGACACAGGCCAGCTGCATCTTATTGCTGAGCTGC CGGTGGTTGCTTATTCCCTGGCTTATCCCTTGGGG GTACTGATTGTGATCTTGAGCATCGCCATCTTTTC TTCAGTGTTTAAGGTTGACCATAACAAGGAGGCAG	SEQ ID NO: 50

-246WO 2017/023818

PCT/US2016/044922

	AAGAGGCTGGGGTAGCGGTCCAAGAACTTAAGGG CCGCCGTATCCGCGTAACTGTAGCTGACTTGCCAG CCCTTGAGAACATTCCTGAGTTGCTTAATTTACAT GTTATCGTCTCGCGTGTAGAGCGCGACGGAGAGC AGTTCATCCCCTTATATGGCGAACATGCACGCATC GGCGATGTACTGACTGTCGTGGGGGCCGACGAGG AACTGAACCGCGCGGAAAAAGCCATCGGAGAGTT AATTGACGGTGATCCTTACTCTAACGTTGAACTGG ACTATCGTCGTATCTTCGTCTCTAATACGGCGGTT GTCGGTACACCCCTGAGCAAATTGCAACCGCTTTT TAAAGATATGCTTATTACTCGCATTCGCCGCGGTG ATACGGATCTGGTAGCTTCCTCGGACATGACGCTT CAATTAGGCGACCGCGTTCGTGTGGTTGCCCCAG CCGAGAAACTTCGTGAAGCGACTCAGTTGCTTGG AGACTCTTACAAAAAGCTGTCCGACTTTAATTTAT TGCCTCTTGCTGCGGGCTTAATGATTGGCGTCCTT GTTGGAATGGTTGAATTCCCACTGCCTGGGGGGT CATCTTTAAAACTTGGCAATGCCGGTGGTCCGTTG GTTGTCGCGCTGTTGCTTGGGATGATCAATCGTAC GGGAAAGTTCGTCTGGCAGATCCCGTACGGAGCA AACTTGGCGTTACGTCAGTTGGGTATCACCCTGTT CTTGGCGGCTATTGGCACTTCCGCGGGAGCTGGG TTTCGCTCAGCTATTAGCGACCCGCAATCTCTGAC CATTATTGGATTTGGTGCGTTGTTAACCTTGTTTA TTAGTATTACCGTCTTGTTCGTTGGGCATAAGTTG ATGAAAATCCCGTTTGGGGAAACGGCGGGTATCT TAGCTGGAACGCAGACCCATCCAGCAGTATTATCA TATGTGTCTGACGCATCTCGCAACGAGTTGCCAGC CATGGGGTACACCTCAGTGTATCCCTTGGCTATGA TTGCGAAAATCCTGGCTGCACAAACACTTTTGTTT CTGTTGATTtaatgaGGGCCCAATAGGCTCCCTATAAGA
GATAGAACTATGCTGACATTCATTGAACTCCTTATT
GGGGTTGTGGTTATTGTGGGTGTAGCTCGCTACAT
CATTAAAGGGTATTCTGCCACTGGCGTGTTATTTG
TCGGTGGCCTGTTATTGCTGATTATCAGTGCCATT
ATGGGGCACAAAGTGTTACCGTCCAGCCAGGCTT
CAACAGGCTACAGCGCCACGGATATCGTTGAATA
CGTTAAAATATTGCTAATGAGCCGCGGCGGCGAC
CTCGGCATGATGATTATGATGCTGTGTGGCTTTGC
CGCTTACATGACCCATATCGGCGCGAATGATATGG
TGGTCAAGCTGGCGTCAAAACCATTGCAGTATATT
AACTCCCCTTACCTTCTGATGATTGCCGCCTATTT
TGTTGCCTGTCTGATGTCACTGGCCGTCTCTTCCG
CAACCGGTCTGGGTGTTTTGCTGATGGCAACCCTG
TTTCCGGTGATGGTAAACGTTGGTATCAGTCGTGG
CGCAGCTGCTGCCATTTGTGCCTCCCCGGCGGCG
ATTATTCTCGCACCGACTTCAGGGGATGTGGTGCT
GGCGGCGCAGGCTTCCGAAATGTCGCTGATTGAC
TTCGCCTTCAAAACAACGCTGCCTATCTCAATTGC

-247WO 2017/023818

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	TGCAATTATCGGCATGGCGATCGCCCACTTCTTCT
GGCAACGTTATCTGGATAAAAAAGAGCACATCTCT
CATGAAATGTTAGATGTCAGTGAAATCACCACCAC
TGCCCCTGCGTTTTATGCCATTTTGCCGTTCACGC
CGATCATCGGAGTACTGATTTTTGACGGCAAATGG
GGTCCGCAATTACACATCATCACTATTCTGGTGAT
TTGTATGCTAATTGCCTCCATTCTGGAGTTCATCC
GCAGCTTTAATACCCAGAAAGTTTTCTCTGGTCTG
GAAGTGGCTTATCGCGGTATGGCAGATGCATTTG
CTAACGTGGTGATGCTGCTGGTTGCCGCTGGGGT
ATTCGCTCAGGGGCTTAGCACCATCGGCTTTATTC
AAAGTCTGATTTCTATCGCTACCTCGTTTGGTTCG
GCGAGTATCATCCTGATGCTGGTATTGGTGATCCT
GACAATGCTGGCGGCAGTCACGACCGGTTCAGGC
AATGCGCCGTTTTATGCGTTTGTTGAGATGATCCC
GAAACTGGCGCACTCCTCCGGCATTAACCCGGCG
TATTTGACTATCCCGATGCTGCAGGCGTCAAACCT
GGGTCGTACCCTATCACCCGTTTCTGGCGTAGTCG
TTGCGGTTGCCGGGATGGCGAAGATCTCGCCGTT
TGAAGTCGTAAAACGCACCTCGGTGCCGGTGCTT
GTTGGTTTGGTGATTGTTATCGTTGCTACAGAGCT
GATGGTGCCAGGAACGGCAGCAGCGGTCACAGGC
AAGTAAGGAATCGACTCCACGTCCCTAGCGTGTGTA GGCAGGAGCAGCAACGAAGTTCCTATACTTTCTAGAGAA
TAGGAACTTC
SucEl (bold) and dcuC (bold underlined) with pAra and RBS (underlined)	ttattcacaacctgccctaaactcgctcggactcgccccggtgcattttttaaatactcgcg agaaatagagttgatcgtcaaaaccgacattgcgaccgacggtggcgataggcatccg ggtggtgctcaaaagcagcttcgcctgactgatgcgctggtcctcgcgccagcttaatac gctaatccctaactgctggcggaacaaatgcgacagacgcgacggcgacaggcagac atgctgtgcgacgctggcgatatcaaaattactgtctgccaggtgatcgctgatgtactga caagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatg cgccgcagtaacaattgctcaagcagatttatcgccagcaattccgaatagcgcccttcc ccttgtccggcattaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcat ccgggcgaaagaaaccggtattggcaaatatcgacggccagttaagccattcatgcca gtaggcgcgcggacgaaagtaaacccactggtgataccattcgtgagcctccggatga cgaccgtagtgatgaatctctccaggcgggaacagcaaaatatcacccggtcggcaga caaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataa cctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggc gttaaacccgccaccagatgggcgttaaacgagtatcccggcagcaggggatcattttg cgcttcagccatACTTTTCATACTCCCGCCATTCAGAGAAGAAA CCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGT CTTTTACTGGCTCTTCTCGCTAACCCAACCGGTAACCCC GCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGC CATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC AGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTT GCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCC AGCCTGACGCTTTTTTTCGCAACTCTCTACTGTTTCTCCA 7ACCCGTTTTTTTGGATGGAGTGAAACGATGTCCTTC	SEQ ID NO: 51

-248WO 2017/023818

PCT/US2016/044922

	CTGGTCGAGAATCAATTGTTAGCACTTGTCGTGAT CATGACCGTCGGGCTTTTACTTGGACGTATCAAAA TCTTTGGTTTCCGTTTGGGTGTGGCCGCCGTGTTG TTCGTCGGCCTTGCTTTAAGCACCATTGAGCCCGA CATTTCGGTTCCATCCCTTATTTACGTGGTTGGCC TTTCGCTTTTTGTGTATACTATCGGTCTGGAAGCT GGCCCCGGTTTTTTTACATCTATGAAGACGACGGG TTTGCGCAATAACGCACTGACGTTAGGTGCCATTA TCGCGACAACAGCACTTGCGTGGGCACTGATTAC CGTCTTGAATATTGATGCCGCCTCAGGAGCTGGTA TGCTTACTGGTGCCTTAACTAATACGCCCGCTATG GCTGCGGTAGTGGATGCACTTCCCTCATTAATTGA TGACACAGGCCAGCTGCATCTTATTGCTGAGCTGC CGGTGGTTGCTTATTCCCTGGCTTATCCCTTGGGG GTACTGATTGTGATCTTGAGCATCGCCATCTTTTC TTCAGTGTTTAAGGTTGACCATAACAAGGAGGCAG AAGAGGCTGGGGTAGCGGTCCAAGAACTTAAGGG CCGCCGTATCCGCGTAACTGTAGCTGACTTGCCAG CCCTTGAGAACATTCCTGAGTTGCTTAATTTACAT GTTATCGTCTCGCGTGTAGAGCGCGACGGAGAGC AGTTCATCCCCTTATATGGCGAACATGCACGCATC GGCGATGTACTGACTGTCGTGGGGGCCGACGAGG AACTGAACCGCGCGGAAAAAGCCATCGGAGAGTT AATTGACGGTGATCCTTACTCTAACGTTGAACTGG ACTATCGTCGTATCTTCGTCTCTAATACGGCGGTT GTCGGTACACCCCTGAGCAAATTGCAACCGCTTTT TAAAGATATGCTTATTACTCGCATTCGCCGCGGTG ATACGGATCTGGTAGCTTCCTCGGACATGACGCTT CAATTAGGCGACCGCGTTCGTGTGGTTGCCCCAG CCGAGAAACTTCGTGAAGCGACTCAGTTGCTTGG AGACTCTTACAAAAAGCTGTCCGACTTTAATTTAT TGCCTCTTGCTGCGGGCTTAATGATTGGCGTCCTT GTTGGAATGGTTGAATTCCCACTGCCTGGGGGGT CATCTTTAAAACTTGGCAATGCCGGTGGTCCGTTG GTTGTCGCGCTGTTGCTTGGGATGATCAATCGTAC GGGAAAGTTCGTCTGGCAGATCCCGTACGGAGCA AACTTGGCGTTACGTCAGTTGGGTATCACCCTGTT CTTGGCGGCTATTGGCACTTCCGCGGGAGCTGGG TTTCGCTCAGCTATTAGCGACCCGCAATCTCTGAC CATTATTGGATTTGGTGCGTTGTTAACCTTGTTTA TTAGTATTACCGTCTTGTTCGTTGGGCATAAGTTG ATGAAAATCCCGTTTGGGGAAACGGCGGGTATCT TAGCTGGAACGCAGACCCATCCAGCAGTATTATCA TATGTGTCTGACGCATCTCGCAACGAGTTGCCAGC CATGGGGTACACCTCAGTGTATCCCTTGGCTATGA TTGCGAAAATCCTGGCTGCACAAACACTTTTGTTT CTGTTGATTtaatgaGGGCCCAATAGGCTCCCTATAAGA
GATAGAACTATGCTGACATTCATTGAACTCCTTATT
GGGGTTGTGGTTATTGTGGGTGTAGCTCGCTACAT

-249WO 2017/023818

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	CATTAAAGGGTATTCTGCCACTGGCGTGTTATTTG
TCGGTGGCCTGTTATTGCTGATTATCAGTGCCATT
ATGGGGCACAAAGTGTTACCGTCCAGCCAGGCTT
CAACAGGCTACAGCGCCACGGATATCGTTGAATA
CGTTAAAATATTGCTAATGAGCCGCGGCGGCGAC
CTCGGCATGATGATTATGATGCTGTGTGGCTTTGC
CGCTTACATGACCCATATCGGCGCGAATGATATGG
TGGTCAAGCTGGCGTCAAAACCATTGCAGTATATT
AACTCCCCTTACCTTCTGATGATTGCCGCCTATTT
TGTTGCCTGTCTGATGTCACTGGCCGTCTCTTCCG
CAACCGGTCTGGGTGTTTTGCTGATGGCAACCCTG
TTTCCGGTGATGGTAAACGTTGGTATCAGTCGTGG
CGCAGCTGCTGCCATTTGTGCCTCCCCGGCGGCG
ATTATTCTCGCACCGACTTCAGGGGATGTGGTGCT
GGCGGCGCAGGCTTCCGAAATGTCGCTGATTGAC
TTCGCCTTCAAAACAACGCTGCCTATCTCAATTGC
TGCAATTATCGGCATGGCGATCGCCCACTTCTTCT
GGCAACGTTATCTGGATAAAAAAGAGCACATCTCT
CATGAAATGTTAGATGTCAGTGAAATCACCACCAC
TGCCCCTGCGTTTTATGCCATTTTGCCGTTCACGC
CGATCATCGGAGTACTGATTTTTGACGGCAAATGG
GGTCCGCAATTACACATCATCACTATTCTGGTGAT
TTGTATGCTAATTGCCTCCATTCTGGAGTTCATCC
GCAGCTTTAATACCCAGAAAGTTTTCTCTGGTCTG
GAAGTGGCTTATCGCGGTATGGCAGATGCATTTG
CTAACGTGGTGATGCTGCTGGTTGCCGCTGGGGT
ATTCGCTCAGGGGCTTAGCACCATCGGCTTTATTC
AAAGTCTGATTTCTATCGCTACCTCGTTTGGTTCG
GCGAGTATCATCCTGATGCTGGTATTGGTGATCCT
GACAATGCTGGCGGCAGTCACGACCGGTTCAGGC
AATGCGCCGTTTTATGCGTTTGTTGAGATGATCCC
GAAACTGGCGCACTCCTCCGGCATTAACCCGGCG
TATTTGACTATCCCGATGCTGCAGGCGTCAAACCT
GGGTCGTACCCTATCACCCGTTTCTGGCGTAGTCG
TTGCGGTTGCCGGGATGGCGAAGATCTCGCCGTT
TGAAGTCGTAAAACGCACCTCGGTGCCGGTGCTT
GTTGGTTTGGTGATTGTTATCGTTGCTACAGAGCT
GATGGTGCCAGGAACGGCAGCAGCGGTCACAGGC
AAGTAA
SucEl (bold) and dcuC (bold underlined) with RBS (underlined)	CCCGTTTTTTTGGATGGAGTGAAACGATGTCCTTCCT	SEQ ID NO: 52
GGTCGAGAATCAATTGTTAGCACTTGTCGTGATCA TGACCGTCGGGCTTTTACTTGGACGTATCAAAATC TTTGGTTTCCGTTTGGGTGTGGCCGCCGTGTTGTT CGTCGGCCTTGCTTTAAGCACCATTGAGCCCGACA TTTCGGTTCCATCCCTTATTTACGTGGTTGGCCTT TCGCTTTTTGTGTATACTATCGGTCTGGAAGCTGG CCCCGGTTTTTTTACATCTATGAAGACGACGGGTT

-250WO 2017/023818

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	TGCGCAATAACGCACTGACGTTAGGTGCCATTATC GCGACAACAGCACTTGCGTGGGCACTGATTACCG TCTTGAATATTGATGCCGCCTCAGGAGCTGGTATG CTTACTGGTGCCTTAACTAATACGCCCGCTATGGC TGCGGTAGTGGATGCACTTCCCTCATTAATTGATG ACACAGGCCAGCTGCATCTTATTGCTGAGCTGCCG GTGGTTGCTTATTCCCTGGCTTATCCCTTGGGGGT ACTGATTGTGATCTTGAGCATCGCCATCTTTTCTT CAGTGTTTAAGGTTGACCATAACAAGGAGGCAGA AGAGGCTGGGGTAGCGGTCCAAGAACTTAAGGGC CGCCGTATCCGCGTAACTGTAGCTGACTTGCCAGC CCTTGAGAACATTCCTGAGTTGCTTAATTTACATG TTATCGTCTCGCGTGTAGAGCGCGACGGAGAGCA GTTCATCCCCTTATATGGCGAACATGCACGCATCG GCGATGTACTGACTGTCGTGGGGGCCGACGAGGA ACTGAACCGCGCGGAAAAAGCCATCGGAGAGTTA ATTGACGGTGATCCTTACTCTAACGTTGAACTGGA CTATCGTCGTATCTTCGTCTCTAATACGGCGGTTG TCGGTACACCCCTGAGCAAATTGCAACCGCTTTTT AAAGATATGCTTATTACTCGCATTCGCCGCGGTGA TACGGATCTGGTAGCTTCCTCGGACATGACGCTTC AATTAGGCGACCGCGTTCGTGTGGTTGCCCCAGC CGAGAAACTTCGTGAAGCGACTCAGTTGCTTGGA GACTCTTACAAAAAGCTGTCCGACTTTAATTTATT GCCTCTTGCTGCGGGCTTAATGATTGGCGTCCTTG TTGGAATGGTTGAATTCCCACTGCCTGGGGGGTC ATCTTTAAAACTTGGCAATGCCGGTGGTCCGTTGG TTGTCGCGCTGTTGCTTGGGATGATCAATCGTACG GGAAAGTTCGTCTGGCAGATCCCGTACGGAGCAA ACTTGGCGTTACGTCAGTTGGGTATCACCCTGTTC TTGGCGGCTATTGGCACTTCCGCGGGAGCTGGGT TTCGCTCAGCTATTAGCGACCCGCAATCTCTGACC ATTATTGGATTTGGTGCGTTGTTAACCTTGTTTAT TAGTATTACCGTCTTGTTCGTTGGGCATAAGTTGA TGAAAATCCCGTTTGGGGAAACGGCGGGTATCTT AGCTGGAACGCAGACCCATCCAGCAGTATTATCAT ATGTGTCTGACGCATCTCGCAACGAGTTGCCAGCC ATGGGGTACACCTCAGTGTATCCCTTGGCTATGAT TGCGAAAATCCTGGCTGCACAAACACTTTTGTTTC TGTTGATTtaatgaGGGCCCAATAGGCTCCCTATAAGAG
ATAGAACTATGCTGACATTCATTGAACTCCTTATTG
GGGTTGTGGTTATTGTGGGTGTAGCTCGCTACATC
ATTAAAGGGTATTCTGCCACTGGCGTGTTATTTGT
CGGTGGCCTGTTATTGCTGATTATCAGTGCCATTA
TGGGGCACAAAGTGTTACCGTCCAGCCAGGCTTC
AACAGGCTACAGCGCCACGGATATCGTTGAATAC
GTTAAAATATTGCTAATGAGCCGCGGCGGCGACC
TCGGCATGATGATTATGATGCTGTGTGGCTTTGCC
GCTTACATGACCCATATCGGCGCGAATGATATGGT

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	GGTCAAGCTGGCGTCAAAACCATTGCAGTATATTA
ACTCCCCTTACCTTCTGATGATTGCCGCCTATTTT
GTTGCCTGTCTGATGTCACTGGCCGTCTCTTCCGC
AACCGGTCTGGGTGTTTTGCTGATGGCAACCCTGT
TTCCGGTGATGGTAAACGTTGGTATCAGTCGTGGC
GCAGCTGCTGCCATTTGTGCCTCCCCGGCGGCGA
TTATTCTCGCACCGACTTCAGGGGATGTGGTGCTG
GCGGCGCAGGCTTCCGAAATGTCGCTGATTGACT
TCGCCTTCAAAACAACGCTGCCTATCTCAATTGCT
GCAATTATCGGCATGGCGATCGCCCACTTCTTCTG
GCAACGTTATCTGGATAAAAAAGAGCACATCTCTC
ATGAAATGTTAGATGTCAGTGAAATCACCACCACT
GCCCCTGCGTTTTATGCCATTTTGCCGTTCACGCC
GATCATCGGAGTACTGATTTTTGACGGCAAATGGG
GTCCGCAATTACACATCATCACTATTCTGGTGATT
TGTATGCTAATTGCCTCCATTCTGGAGTTCATCCG
CAGCTTTAATACCCAGAAAGTTTTCTCTGGTCTGG
AAGTGGCTTATCGCGGTATGGCAGATGCATTTGCT
AACGTGGTGATGCTGCTGGTTGCCGCTGGGGTAT
TCGCTCAGGGGCTTAGCACCATCGGCTTTATTCAA
AGTCTGATTTCTATCGCTACCTCGTTTGGTTCGGC
GAGTATCATCCTGATGCTGGTATTGGTGATCCTGA
CAATGCTGGCGGCAGTCACGACCGGTTCAGGCAA
TGCGCCGTTTTATGCGTTTGTTGAGATGATCCCGA
AACTGGCGCACTCCTCCGGCATTAACCCGGCGTAT
TTGACTATCCCGATGCTGCAGGCGTCAAACCTGG
GTCGTACCCTATCACCCGTTTCTGGCGTAGTCGTT
GCGGTTGCCGGGATGGCGAAGATCTCGCCGTTTG
AAGTCGTAAAACGCACCTCGGTGCCGGTGCTTGTT
GGTTTGGTGATTGTTATCGTTGCTACAGAGCTGAT
GGTGCCAGGAACGGCAGCAGCGGTCACAGGCAAG
TAA

Example 14 Activity of the 2-Methyl-Citrate Pathway [0554] To determine the suitability of 2-methyl citrate pathway for propionate consumption by the genetically engineered bacteria, a circuit in which the prpB, prpC, prpD, and prpE genes are expressed under the control of an inducible promoter is generated. 2methyl citrate pathway sequences are shown in Table 16.

Table 16. 2-Methyl Citrate Pathway Circuit Sequences

Description

Sequence

SEQ ID NO

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Construct comprising TetR (reverse orientation, lowercase) and a prpBCDE gene cassette under the Ptet promoter (italics) (as shown in FIG. 20); ribosome binding sites are underlined ;; coding regions in bold underline	ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaag gccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagctt gtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttctttagcg acttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcg ctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaaggct aattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgtactt ttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgt aaaaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgccta tctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgcc aatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgtta aaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttactt ttatctaatctagacatcat TAA TTC CTAA ΤΤΤΠGTTGA CACT CTATCATTGATAGAGTTATTTTACCACTCCCTATCA GTGATAGAGAAAAGTGAAATGTCTCTACACTCT CCAGGTAAAGCGTTTCGCGCTGCACTTAGC	SEQ ID NO: 53
AAAGAAACCCCGTTGCAAATTGTTGGCACC
ATCAACGCTAACCATGCGCTGCTGGCGCAG
CGTGCCGGATATCAGGCGATTTATCTCTCCG
GCGGTGGCGTGGCGGCAGGATCGCTGGGG
CTGCCCGATCTCGGTATTTCTACTCTTGATG
ACGTGCTGACAGATATTCGCCGTATCACCG
ACGTTTGTTCGCTGCCGCTGCTGGTGGATG
CGGATATCGGTTTTGGTTCTTCAGCCTTTAA
CGTGGCGCGTACGGTGAAATCAATGATTAA
AGCCGGTGCGGCAGGATTGCATATTGAAGA
TCAGGTTGGTGCGAAACGCTGCGGTCATCG
TCCGAATAAAGCGATCGTCTCGAAAGAAGA
GATGGTGGATCGGATCCGCGCGGCGGTGGA
TGCGAAAACCGATCCTGATTTTGTGATCATG
GCGCGCACCGATGCGCTGGCGGTAGAGGGG
CTGGATGCGGCGATCGAGCGTGCGCAGGCC
TATGTTGAAGCGGGTGCCGAAATGCTGTTC
CCGGAGGCGATTACCGAACTCGCCATGTAT
CGCCAGTTTGCCGATGCGGTGCAGGTGCCG
ATCCTCTCCAACATTACCGAATTTGGCGCAA
CACCGCTGTTTACCACCGACGAATTACGCA
GCGCCCATGTCGCAATGGCGCTCTACCCGC
TTTCAGCGTTTCGCGCCATGAACCGCGCCG
CTGAACATGTCTATAACATCCTGCGTCAGGA
AGGCACACAGAAAAGCGTCATCGACACCAT
GCAGACCCGCAACGAGCTGTACGAAAGCAT
CAACTACTACCAGTACGAAGAGAAGCTCGA
CGACCTGTTTGCCCGTGGTCAGGTGAAATA
A AAACGCCCGTTGGTTGTATTCGACAACCGATG CCTGATGCGCCGCTGACGCGACTTATCAGGCC TACGAGGTGAACTGAACTGTAGGTCGGATAAG ACGCATAGCGTCGCATCCGACAACAATCTCGA CCCTACAAATGATAACAATGACGAGGACAATA

-253WO 2017/023818

PCT/US2016/044922

	TGAGCGACACAACGATCCTGCAAAACAGTA
CCCATGTCATTAAACCGAAAAAATCGGTGG
CACTTTCCGGCGTTCCGGCGGGCAATACGG
CGCTCTGCACCGTGGGTAAAAGCGGCAACG
ACCTGCATTACCGTGGCTACGATATTCTTGA
TCTGGCGGAACATTGTGAATTTGAAGAAGT
GGCGCACCTGCTGATCCACGGCAAACTGCC
AACCCGTGACGAACTCGCCGCCTACAAAAC
GAAACTGAAAGCCCTGCGTGGTTTACCGGC
TAACGTGCGTACCGTGCTGGAAGCCTTACC
GGCGGCGTCACACCCGATGGATGTTATGCG
CACCGGCGTTTCCGCGCTCGGCTGCACGCT
GCCAGAAAAAGAGGGGCACACCGTTTCTGG
TGCGCGGGATATTGCCGACAAACTGCTGGC
GTCACTTAGTTCGATTCTTCTCTACTGGTAT
CACTACAGCCACAACGGCGAACGCATCCAG
CCGGAAACTGATGACGACTCTATCGGCGGT
CACTTCCTGCATCTGCTGCACGGCGAAAAG
CCGTCGCAAAGCTGGGAAAAGGCGATGCAT
ATCTCGCTGGTGCTGTACGCCGAACACGAG
TTTAACGCTTCCACCTTTACCAGCCGGGTGA
TTGCGGGCACTGGCTCTGATATGTATTCCGC
CATTATTGGCGCGATTGGCGCACTGCGCGG
GCCGAAACACGGCGGGGCGAATGAAGTGTC
GCTGGAGATCCAGCAACGCTACGAAACGCC
GGGCGAAGCCGAAGCCGATATCCGCAAGCG
GGTGGAAAACAAAGAAGTGGTCATTGGTTT
TGGGCATCCGGTTTATACCATCGCCGACCC
GCGTCATCAGGTGATCAAACGTGTGGCGAA
GCAGCTCTCGCAGGAAGGCGGCTCGCTGAA
GATGTACAACATCGCCGATCGCCTGGAAAC
GGTGATGTGGGAGAGCAAAAAGATGTTCCC
CAATCTCGACTGGTTCTCCGCTGTTTCCTAC
AACATGATGGGTGTTCCCACCGAGATGTTC
ACACCACTGTTTGTTATCGCCCGCGTCACTG
GCTGGGCGGCGCACATTATCGAACAACGTC
AGGACAACAAAATTATCCGTCCTTCCGCCAA
TTATGTTGGACCGGAAGACCGCCAGTTTGT
CGCGCTGGATAAGCGCCAGTAA ACCTCTACGAATAACAATAAGGAAACGTACCC AATGTCAGCTCAAATCAACAACATCCGCCCG
GAATTTGATCGTGAAATCGTTGATATCGTCG
ATTACGTGATGAACTACGAAATCAGCTCCAG
AGTAGCCTACGACACCGCTCATTACTGCCTG
CTTGACACGCTCGGCTGCGGTCTGGAAGCT
CTCGAATATCCGGCCTGTAAAAAACTGCTG
GGGCCAATTGTCCCCGGCACCGTCGTACCC
AACGGCGTGCGCGTTCCCGGAACTCAGTTT
CAGCTCGACCCCGTCCAGGCGGCATTTAAC

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	ATTGGCGCGATGATCCGTTGGCTCGATTTCA
ACGATACCTGGCTGGCGGCGGAGTGGGGGC
ATCCTTCCGACAACCTCGGCGGCATTCTGG
CAACGGCGGACTGGCTTTCGCGCAACGCGA
TCGCCAGCGGCAAAGCGCCGTTGACCATGA
AACAGGTGCTGACCGGAATGATCAAAGCCC
ATGAAATTCAGGGCTGCATCGCGCTGGAAA
ACTCCTTTAACCGCGTTGGTCTCGACCACGT
TCTGTTAGTGAAAGTGGCTTCCACCGCCGT
GGTCGCCGAAATGCTCGGCCTGACCCGCGA
GGAAATTCTCAACGCCGTTTCGCTGGCATG
GGTAGACGGACAGTCGCTGCGCACTTATCG
TCATGCACCGAACACCGGTACGCGTAAATC
CTGGGCGGCGGGCGATGCTACATCCCGCGC
GGTACGTCTGGCGCTGATGGCGAAAACGGG
CGAAATGGGTTACCCGTCAGCCCTGACCGC
GCCGGTGTGGGGTTTCTACGACGTCTCCTTT
AAAGGTGAGTCATTCCGCTTCCAGCGTCCG
TACGGTTCCTACGTCATGGAAAATGTGCTGT
TCAAAATCTCCTTCCCGGCGGAGTTCCACTC
CCAGACGGCAGTTGAAGCGGCGATGACGCT
CTATGAACAGATGCAGGCAGCAGGCAAAAC
GGCGGCAGATATCGAAAAAGTGACCATTCG
CACCCACGAAGCCTGTATTCGCATCATCGAC
AAAAAAGGGCCGCTCAATAACCCGGCAGAC
CGCGACCACTGCATTCAGTACATGGTGGCG
ATCCCGCTGCTGTTCGGACGCTTAACGGCG
GCAGATTACGAGGACAACGTTGCGCAAGAT
AAACGCATCGACGCCCTGCGCGAGAAGATC
AATTGCTTTGAAGATCCGGCGTTTACCGCTG
ACTACCACGACCCGGAAAAACGCGCCATCG
CCAATGCCATAACCCTTGAGTTCACCGACG
GCACACGATTTGAAGAAGTGGTGGTGGAGT
ACCCAATTGGTCATGCTCGCCGCCGTCAGG
ATGGCATTCCGAAGCTGGTCGATAAATTCAA
AATCAATCTCGCGCGCCAGTTCCCGACTCG
CCAGCAGCAGCGCATTCTGGAGGTTTCTCT
CGACAGAACTCGCCTGGAACAGATGCCGGT
CAATGAGTATCTCGACCTGTACGTCATTTAA
GTAAACGGCGGTAAGGCGTAAGTTCAACAGGA GAGCATTATGTCTTTTAGCGAATTTTATCAG CGTTCGATTAACGAACCGGAGAAGTTCTGG
GCCGAGCAGGCCCGGCGTATTGACTGGCAG
ACGCCCTTTACGCAAACGCTCGACCACAGC
AACCCGCCGTTTGCCCGTTGGTTTTGTGAAG
GCCGAACCAACTTGTGTCACAACGCTATCG
ACCGCTGGCTGGAGAAACAGCCAGAGGCGC
TGGCATTGATTGCCGTCTCTTCGGAAACAGA
GGAAGAGCGTACCTTTACCTTCCGCCAGTTA

-255WO 2017/023818

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	CATGACGAAGTGAATGCGGTGGCGTCAATG
CTGCGCTCACTGGGCGTGCAGCGTGGCGAT
CGGGTGCTGGTGTATATGCCGATGATTGCC
GAAGCGCATATTACCCTGCTGGCCTGCGCG
CGCATTGGTGCTATTCACTCGGTGGTGTTTG
GGGGATTTGCTTCGCACAGCGTGGCAACGC
GAATTGATGACGCTAAACCGGTGCTGATTG
TCTCGGCTGATGCCGGGGCGCGCGGCGGTA
AAATCATTCCGTATAAAAAATTGCTCGACGA
TGCGATAAGTCAGGCACAGCATCAGCCGCG
TCACGTTTTACTGGTGGATCGCGGGCTGGC
GAAAATGGCGCGCGTTAGCGGGCGGGATGT
CGATTTCGCGTCGTTGCGCCATCAACACATC
GGCGCGCGGGTGCCGGTGGCATGGCTGGAA
TCCAACGAAACCTCCTGCATTCTCTACACCT
CCGGCACGACCGGCAAACCTAAAGGTGTGC
AGCGTGATGTCGGCGGATATGCGGTGGCGC
TGGCGACCTCGATGGACACCATTTTTGGCG
GCAAAGCGGGCGGCGTGTTCTTTTGTGCTT
CGGATATCGGCTGGGTGGTAGGGCATTCGT
ATATCGTTTACGCGCCGCTGCTGGCGGGGA
TGGCGACTATCGTTTACGAAGGATTGCCGA
CCTGGCCGGACTGCGGCGTGTGGTGGAAAA
TTGTCGAGAAATATCAGGTTAGCCGCATGTT
CTCAGCGCCGACCGCCATTCGCGTGCTGAA
AAAATTCCCTACCGCTGAAATTCGCAAACAC
GATCTTTCGTCGCTGGAAGTGCTCTATCTGG
CTGGAGAACCGCTGGACGAGCCGACCGCCA
GTTGGGTGAGCAATACGCTGGATGTGCCGG
TCATCGACAACTACTGGCAGACCGAATCCG
GCTGGCCGATTATGGCGATTGCTCGCGGTC
TGGATGACAGACCGACGCGTCTGGGAAGCC
CCGGCGTGCCGATGTATGGCTATAACGTGC
AGTTGCTCAATGAAGTCACCGGCGAACCGT
GTGGCGTCAATGAGAAAGGGATGCTGGTAG
TGGAGGGGCCATTGCCGCCAGGCTGTATTC
AAACCATCTGGGGCGACGACGACCGCTTTG
TGAAGACGTACTGGTCGCTGTTTTCCCGTCC
GGTGTACGCCACTTTTGACTGGGGCATCCG
CGATGCTGACGGTTATCACTTTATTCTCGGG
CGCACTGACGATGTGATTAACGTTGCCGGA
CATCGGCTGGGTACGCGTGAGATTGAAGAG
AGTATCTCCAGTCATCCGGGCGTTGCCGAA
GTGGCGGTGGTTGGGGTGAAAGATGCGCTG
AAAGGGCAGGTGGCGGTGGCGTTTGTCATT
CCGAAAGAGAGCGACAGTCTGGAAGACCGT
GAGGTGGCGCACTCGCAAGAGAAGGCGATT
ATGGCGCTGGTGGACAGCCAGATTGGCAAC
TTTGGCCGCCCGGCGCACGTCTGGTTTGTC

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	TCGCAATTGCCAAAAACGCGATCCGGAAAA
ATGCTGCGCCGCACGATCCAGGCGATTTGC
GAAGGACGCGATCCTGGGGATCTGACGACC
ATTGATGATCCGGCGTCGTTGGATCAGATC
CGCCAGGCGATGGAAGAGTAGGTCGGATAA
GGCGCTCGCGCCGCATCCGACACCGTGCGCAG ATGCCTGATGCGACGCTGACGCGTCTTATCATG CCTCGCTCTCGAGTCCCGTCAAGTCAGCGTAAT GCTCTGCCAGTGTTACAACCAATTAACCAATTC TGAT
Construct comprising a prpBCDE gene cassette under the control of the Ptet promoter (italics) (as shown in FIG. 20) ribosome binding sites are underlined ; coding regions in bold underlined;.	GAGTTATTTTACCACTCCCTATCAGTGATAGAGAA AAGTGAA ATGTCTCTACACTCTCCAGGTAAAGCGTTTC	SEQ ID NO: 54
GCGCTGCACTTAGCAAAGAAACCCCGTTGC
AAATTGTTGGCACCATCAACGCTAACCATGC
GCTGCTGGCGCAGCGTGCCGGATATCAGGC
GATTTATCTCTCCGGCGGTGGCGTGGCGGC
AGGATCGCTGGGGCTGCCCGATCTCGGTAT
TTCTACTCTTGATGACGTGCTGACAGATATT
CGCCGTATCACCGACGTTTGTTCGCTGCCG
CTGCTGGTGGATGCGGATATCGGTTTTGGT
TCTTCAGCCTTTAACGTGGCGCGTACGGTG
AAATCAATGATTAAAGCCGGTGCGGCAGGA
TTGCATATTGAAGATCAGGTTGGTGCGAAA
CGCTGCGGTCATCGTCCGAATAAAGCGATC
GTCTCGAAAGAAGAGATGGTGGATCGGATC
CGCGCGGCGGTGGATGCGAAAACCGATCCT
GATTTTGTGATCATGGCGCGCACCGATGCG
CTGGCGGTAGAGGGGCTGGATGCGGCGATC
GAGCGTGCGCAGGCCTATGTTGAAGCGGGT
GCCGAAATGCTGTTCCCGGAGGCGATTACC
GAACTCGCCATGTATCGCCAGTTTGCCGAT
GCGGTGCAGGTGCCGATCCTCTCCAACATT
ACCGAATTTGGCGCAACACCGCTGTTTACCA
CCGACGAATTACGCAGCGCCCATGTCGCAA
TGGCGCTCTACCCGCTTTCAGCGTTTCGCGC
CATGAACCGCGCCGCTGAACATGTCTATAA
CATCCTGCGTCAGGAAGGCACACAGAAAAG
CGTCATCGACACCATGCAGACCCGCAACGA
GCTGTACGAAAGCATCAACTACTACCAGTAC
GAAGAGAAGCTCGACGACCTGTTTGCCCGT
GGTCAGGTGAAATAA AAACGCCCGTTGGTTGTATTCGACAACCGATG CCTGATGCGCCGCTGACGCGACTTATCAGGCC TACGAGGTGAACTGAACTGTAGGTCGGATAAG ACGCATAGCGTCGCATCCGACAACAATCTCGA CCCTACAAATGATAACAATGACGAGGACAATA

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	TCGCAATTGCCAAAAACGCGATCCGGAAAA
ATGCTGCGCCGCACGATCCAGGCGATTTGC
GAAGGACGCGATCCTGGGGATCTGACGACC
ATTGATGATCCGGCGTCGTTGGATCAGATC
CGCCAGGCGATGGAAGAGTAG
Construct comprising a prpBCDE gene cassette; (as shown in FIG. 20) ribosome binding sites are underlined; coding region in bold	ATGTCTCTACACTCTCCAGGTAAAGCGTTTC	SEQ ID NO: 55
GCGCTGCACTTAGCAAAGAAACCCCGTTGC
AAATTGTTGGCACCATCAACGCTAACCATGC
GCTGCTGGCGCAGCGTGCCGGATATCAGGC
GATTTATCTCTCCGGCGGTGGCGTGGCGGC
AGGATCGCTGGGGCTGCCCGATCTCGGTAT
TTCTACTCTTGATGACGTGCTGACAGATATT
CGCCGTATCACCGACGTTTGTTCGCTGCCG
CTGCTGGTGGATGCGGATATCGGTTTTGGT
TCTTCAGCCTTTAACGTGGCGCGTACGGTG
AAATCAATGATTAAAGCCGGTGCGGCAGGA
TTGCATATTGAAGATCAGGTTGGTGCGAAA
CGCTGCGGTCATCGTCCGAATAAAGCGATC
GTCTCGAAAGAAGAGATGGTGGATCGGATC
CGCGCGGCGGTGGATGCGAAAACCGATCCT
GATTTTGTGATCATGGCGCGCACCGATGCG
CTGGCGGTAGAGGGGCTGGATGCGGCGATC
GAGCGTGCGCAGGCCTATGTTGAAGCGGGT
GCCGAAATGCTGTTCCCGGAGGCGATTACC
GAACTCGCCATGTATCGCCAGTTTGCCGAT
GCGGTGCAGGTGCCGATCCTCTCCAACATT
ACCGAATTTGGCGCAACACCGCTGTTTACCA
CCGACGAATTACGCAGCGCCCATGTCGCAA
TGGCGCTCTACCCGCTTTCAGCGTTTCGCGC
CATGAACCGCGCCGCTGAACATGTCTATAA
CATCCTGCGTCAGGAAGGCACACAGAAAAG
CGTCATCGACACCATGCAGACCCGCAACGA
GCTGTACGAAAGCATCAACTACTACCAGTAC
GAAGAGAAGCTCGACGACCTGTTTGCCCGT
GGTCAGGTGAAATAA AAACGCCCGTTGGTTGTATTCGACAACCGATG CCTGATGCGCCGCTGACGCGACTTATCAGGCC TACGAGGTGAACTGAACTGTAGGTCGGATAAG ACGCATAGCGTCGCATCCGACAACAATCTCGA CCCTACAAATGATAACAATGACGAGGACAATA
TGAGCGACACAACGATCCTGCAAAACAGTA
CCCATGTCATTAAACCGAAAAAATCGGTGG
CACTTTCCGGCGTTCCGGCGGGCAATACGG
CGCTCTGCACCGTGGGTAAAAGCGGCAACG
ACCTGCATTACCGTGGCTACGATATTCTTGA
TCTGGCGGAACATTGTGAATTTGAAGAAGT
GGCGCACCTGCTGATCCACGGCAAACTGCC

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	AACCCGTGACGAACTCGCCGCCTACAAAAC
GAAACTGAAAGCCCTGCGTGGTTTACCGGC
TAACGTGCGTACCGTGCTGGAAGCCTTACC
GGCGGCGTCACACCCGATGGATGTTATGCG
CACCGGCGTTTCCGCGCTCGGCTGCACGCT
GCCAGAAAAAGAGGGGCACACCGTTTCTGG
TGCGCGGGATATTGCCGACAAACTGCTGGC
GTCACTTAGTTCGATTCTTCTCTACTGGTAT
CACTACAGCCACAACGGCGAACGCATCCAG
CCGGAAACTGATGACGACTCTATCGGCGGT
CACTTCCTGCATCTGCTGCACGGCGAAAAG
CCGTCGCAAAGCTGGGAAAAGGCGATGCAT
ATCTCGCTGGTGCTGTACGCCGAACACGAG
TTTAACGCTTCCACCTTTACCAGCCGGGTGA
TTGCGGGCACTGGCTCTGATATGTATTCCGC
CATTATTGGCGCGATTGGCGCACTGCGCGG
GCCGAAACACGGCGGGGCGAATGAAGTGTC
GCTGGAGATCCAGCAACGCTACGAAACGCC
GGGCGAAGCCGAAGCCGATATCCGCAAGCG
GGTGGAAAACAAAGAAGTGGTCATTGGTTT
TGGGCATCCGGTTTATACCATCGCCGACCC
GCGTCATCAGGTGATCAAACGTGTGGCGAA
GCAGCTCTCGCAGGAAGGCGGCTCGCTGAA
GATGTACAACATCGCCGATCGCCTGGAAAC
GGTGATGTGGGAGAGCAAAAAGATGTTCCC
CAATCTCGACTGGTTCTCCGCTGTTTCCTAC
AACATGATGGGTGTTCCCACCGAGATGTTC
ACACCACTGTTTGTTATCGCCCGCGTCACTG
GCTGGGCGGCGCACATTATCGAACAACGTC
AGGACAACAAAATTATCCGTCCTTCCGCCAA
TTATGTTGGACCGGAAGACCGCCAGTTTGT
CGCGCTGGATAAGCGCCAGTAA ACCTCTACGAATAACAATAAGGAAACGTACCC
AATGTCAGCTCAAATCAACAACATCCGCCCG
GAATTTGATCGTGAAATCGTTGATATCGTCG
ATTACGTGATGAACTACGAAATCAGCTCCAG
AGTAGCCTACGACACCGCTCATTACTGCCTG
CTTGACACGCTCGGCTGCGGTCTGGAAGCT
CTCGAATATCCGGCCTGTAAAAAACTGCTG
GGGCCAATTGTCCCCGGCACCGTCGTACCC
AACGGCGTGCGCGTTCCCGGAACTCAGTTT
CAGCTCGACCCCGTCCAGGCGGCATTTAAC
ATTGGCGCGATGATCCGTTGGCTCGATTTCA
ACGATACCTGGCTGGCGGCGGAGTGGGGGC
ATCCTTCCGACAACCTCGGCGGCATTCTGG
CAACGGCGGACTGGCTTTCGCGCAACGCGA
TCGCCAGCGGCAAAGCGCCGTTGACCATGA
AACAGGTGCTGACCGGAATGATCAAAGCCC
ATGAAATTCAGGGCTGCATCGCGCTGGAAA

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	ACTCCTTTAACCGCGTTGGTCTCGACCACGT
TCTGTTAGTGAAAGTGGCTTCCACCGCCGT
GGTCGCCGAAATGCTCGGCCTGACCCGCGA
GGAAATTCTCAACGCCGTTTCGCTGGCATG
GGTAGACGGACAGTCGCTGCGCACTTATCG
TCATGCACCGAACACCGGTACGCGTAAATC
CTGGGCGGCGGGCGATGCTACATCCCGCGC
GGTACGTCTGGCGCTGATGGCGAAAACGGG
CGAAATGGGTTACCCGTCAGCCCTGACCGC
GCCGGTGTGGGGTTTCTACGACGTCTCCTTT
AAAGGTGAGTCATTCCGCTTCCAGCGTCCG
TACGGTTCCTACGTCATGGAAAATGTGCTGT
TCAAAATCTCCTTCCCGGCGGAGTTCCACTC
CCAGACGGCAGTTGAAGCGGCGATGACGCT
CTATGAACAGATGCAGGCAGCAGGCAAAAC
GGCGGCAGATATCGAAAAAGTGACCATTCG
CACCCACGAAGCCTGTATTCGCATCATCGAC
AAAAAAGGGCCGCTCAATAACCCGGCAGAC
CGCGACCACTGCATTCAGTACATGGTGGCG
ATCCCGCTGCTGTTCGGACGCTTAACGGCG
GCAGATTACGAGGACAACGTTGCGCAAGAT
AAACGCATCGACGCCCTGCGCGAGAAGATC
AATTGCTTTGAAGATCCGGCGTTTACCGCTG
ACTACCACGACCCGGAAAAACGCGCCATCG
CCAATGCCATAACCCTTGAGTTCACCGACG
GCACACGATTTGAAGAAGTGGTGGTGGAGT
ACCCAATTGGTCATGCTCGCCGCCGTCAGG
ATGGCATTCCGAAGCTGGTCGATAAATTCAA
AATCAATCTCGCGCGCCAGTTCCCGACTCG
CCAGCAGCAGCGCATTCTGGAGGTTTCTCT
CGACAGAACTCGCCTGGAACAGATGCCGGT
CAATGAGTATCTCGACCTGTACGTCATTTAA
GTAAACGGCGGTAAGGCGTAAGTTCAACAGGA
GAGCATTATGTCTTTTAGCGAATTTTATCAG
CGTTCGATTAACGAACCGGAGAAGTTCTGG
GCCGAGCAGGCCCGGCGTATTGACTGGCAG
ACGCCCTTTACGCAAACGCTCGACCACAGC
AACCCGCCGTTTGCCCGTTGGTTTTGTGAAG
GCCGAACCAACTTGTGTCACAACGCTATCG
ACCGCTGGCTGGAGAAACAGCCAGAGGCGC
TGGCATTGATTGCCGTCTCTTCGGAAACAGA
GGAAGAGCGTACCTTTACCTTCCGCCAGTTA
CATGACGAAGTGAATGCGGTGGCGTCAATG
CTGCGCTCACTGGGCGTGCAGCGTGGCGAT
CGGGTGCTGGTGTATATGCCGATGATTGCC
GAAGCGCATATTACCCTGCTGGCCTGCGCG
CGCATTGGTGCTATTCACTCGGTGGTGTTTG
GGGGATTTGCTTCGCACAGCGTGGCAACGC
GAATTGATGACGCTAAACCGGTGCTGATTG

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	TCTCGGCTGATGCCGGGGCGCGCGGCGGTA
AAATCATTCCGTATAAAAAATTGCTCGACGA
TGCGATAAGTCAGGCACAGCATCAGCCGCG
TCACGTTTTACTGGTGGATCGCGGGCTGGC
GAAAATGGCGCGCGTTAGCGGGCGGGATGT
CGATTTCGCGTCGTTGCGCCATCAACACATC
GGCGCGCGGGTGCCGGTGGCATGGCTGGAA
TCCAACGAAACCTCCTGCATTCTCTACACCT
CCGGCACGACCGGCAAACCTAAAGGTGTGC
AGCGTGATGTCGGCGGATATGCGGTGGCGC
TGGCGACCTCGATGGACACCATTTTTGGCG
GCAAAGCGGGCGGCGTGTTCTTTTGTGCTT
CGGATATCGGCTGGGTGGTAGGGCATTCGT
ATATCGTTTACGCGCCGCTGCTGGCGGGGA
TGGCGACTATCGTTTACGAAGGATTGCCGA
CCTGGCCGGACTGCGGCGTGTGGTGGAAAA
TTGTCGAGAAATATCAGGTTAGCCGCATGTT
CTCAGCGCCGACCGCCATTCGCGTGCTGAA
AAAATTCCCTACCGCTGAAATTCGCAAACAC
GATCTTTCGTCGCTGGAAGTGCTCTATCTGG
CTGGAGAACCGCTGGACGAGCCGACCGCCA
GTTGGGTGAGCAATACGCTGGATGTGCCGG
TCATCGACAACTACTGGCAGACCGAATCCG
GCTGGCCGATTATGGCGATTGCTCGCGGTC
TGGATGACAGACCGACGCGTCTGGGAAGCC
CCGGCGTGCCGATGTATGGCTATAACGTGC
AGTTGCTCAATGAAGTCACCGGCGAACCGT
GTGGCGTCAATGAGAAAGGGATGCTGGTAG
TGGAGGGGCCATTGCCGCCAGGCTGTATTC
AAACCATCTGGGGCGACGACGACCGCTTTG
TGAAGACGTACTGGTCGCTGTTTTCCCGTCC
GGTGTACGCCACTTTTGACTGGGGCATCCG
CGATGCTGACGGTTATCACTTTATTCTCGGG
CGCACTGACGATGTGATTAACGTTGCCGGA
CATCGGCTGGGTACGCGTGAGATTGAAGAG
AGTATCTCCAGTCATCCGGGCGTTGCCGAA
GTGGCGGTGGTTGGGGTGAAAGATGCGCTG
AAAGGGCAGGTGGCGGTGGCGTTTGTCATT
CCGAAAGAGAGCGACAGTCTGGAAGACCGT
GAGGTGGCGCACTCGCAAGAGAAGGCGATT
ATGGCGCTGGTGGACAGCCAGATTGGCAAC
TTTGGCCGCCCGGCGCACGTCTGGTTTGTC
TCGCAATTGCCAAAAACGCGATCCGGAAAA
ATGCTGCGCCGCACGATCCAGGCGATTTGC
GAAGGACGCGATCCTGGGGATCTGACGACC
ATTGATGATCCGGCGTCGTTGGATCAGATC
CGCCAGGCGATGGAAGAGTAG
prpB sequence	ATGTCTCTACACTCTCCAGGTAAAGCGTTTCGC	SEQ ID

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(comprised in the prpBCDE construct shown in FIG. 20)	GCTGCACTTAGCAAAGAAACCCCGTTGCAAAT TGTTGGCACCATCAACGCTAACCATGCGCTGCT GGCGCAGCGTGCCGGATATCAGGCGATTTATC TCTCCGGCGGTGGCGTGGCGGCAGGATCGCTG GGGCTGCCCGATCTCGGTATTTCTACTCTTGAT GACGTGCTGACAGATATTCGCCGTATCACCGA CGTTTGTTCGCTGCCGCTGCTGGTGGATGCGGA TATCGGTTTTGGTTCTTCAGCCTTTAACGTGGC GCGTACGGTGAAATCAATGATTAAAGCCGGTG CGGCAGGATTGCATATTGAAGATCAGGTTGGT GCGAAACGCTGCGGTCATCGTCCGAATAAAGC GATCGTCTCGAAAGAAGAGATGGTGGATCGGA TCCGCGCGGCGGTGGATGCGAAAACCGATCCT GATTTTGTGATCATGGCGCGCACCGATGCGCT GGCGGTAGAGGGGCTGGATGCGGCGATCGAGC GTGCGCAGGCCTATGTTGAAGCGGGTGCCGAA ATGCTGTTCCCGGAGGCGATTACCGAACTCGC CATGTATCGCCAGTTTGCCGATGCGGTGCAGG TGCCGATCCTCTCCAACATTACCGAATTTGGCG CAACACCGCTGTTTACCACCGACGAATTACGC AGCGCCCATGTCGCAATGGCGCTCTACCCGCTT TCAGCGTTTCGCGCCATGAACCGCGCCGCTGA ACATGTCTATAACATCCTGCGTCAGGAAGGCA CACAGAAAAGCGTCATCGACACCATGCAGACC CGCAACGAGCTGTACGAAAGCATCAACTACTA CCAGTACGAAGAGAAGCTCGACGACCTGTTTG CCCGTGGTCAGGTGAAATAA	NO: 56
prpC sequence (comprised in the prpBCDE construct shown in FIG. 20)	ATGAGCGACACAACGATCCTGCAAAACAGTAC CCATGTCATTAAACCGAAAAAATCGGTGGCAC TTTCCGGCGTTCCGGCGGGCAATACGGCGCTCT GCACCGTGGGTAAAAGCGGCAACGACCTGCAT TACCGTGGCTACGATATTCTTGATCTGGCGGAA CATTGTGAATTTGAAGAAGTGGCGCACCTGCT GATCCACGGCAAACTGCCAACCCGTGACGAAC TCGCCGCCTACAAAACGAAACTGAAAGCCCTG CGTGGTTTACCGGCTAACGTGCGTACCGTGCTG GAAGCCTTACCGGCGGCGTCACACCCGATGGA TGTTATGCGCACCGGCGTTTCCGCGCTCGGCTG CACGCTGCCAGAAAAAGAGGGGCACACCGTTT CTGGTGCGCGGGATATTGCCGACAAACTGCTG GCGTCACTTAGTTCGATTCTTCTCTACTGGTAT CACTACAGCCACAACGGCGAACGCATCCAGCC GGAAACTGATGACGACTCTATCGGCGGTCACT TCCTGCATCTGCTGCACGGCGAAAAGCCGTCG CAAAGCTGGGAAAAGGCGATGCATATCTCGCT GGTGCTGTACGCCGAACACGAGTTTAACGCTT CCACCTTTACCAGCCGGGTGATTGCGGGCACT GGCTCTGATATGTATTCCGCCATTATTGGCGCG	SEQ ID NO: 57

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	ATTGGCGCACTGCGCGGGCCGAAACACGGCGG GGCGAATGAAGTGTCGCTGGAGATCCAGCAAC GCTACGAAACGCCGGGCGAAGCCGAAGCCGAT ATCCGCAAGCGGGTGGAAAACAAAGAAGTGG TCATTGGTTTTGGGCATCCGGTTTATACCATCG CCGACCCGCGTCATCAGGTGATCAAACGTGTG GCGAAGCAGCTCTCGCAGGAAGGCGGCTCGCT GAAGATGTACAACATCGCCGATCGCCTGGAAA CGGTGATGTGGGAGAGCAAAAAGATGTTCCCC AATCTCGACTGGTTCTCCGCTGTTTCCTACAAC ATGATGGGTGTTCCCACCGAGATGTTCACACC ACTGTTTGTTATCGCCCGCGTCACTGGCTGGGC GGCGCACATTATCGAACAACGTCAGGACAACA AAATTATCCGTCCTTCCGCCAATTATGTTGGAC CGGAAGACCGCCAGTTTGTCGCGCTGGATAAG CGCCAGTAA
prpD sequence (comprised in the prpBCDE construct shown in FIG. 20)	ATGTCAGCTCAAATCAACAACATCCGCCCGGA ATTTGATCGTGAAATCGTTGATATCGTCGATTA CGTGATGAACTACGAAATCAGCTCCAGAGTAG CCTACGACACCGCTCATTACTGCCTGCTTGACA CGCTCGGCTGCGGTCTGGAAGCTCTCGAATAT CCGGCCTGTAAAAAACTGCTGGGGCCAATTGT CCCCGGCACCGTCGTACCCAACGGCGTGCGCG TTCCCGGAACTCAGTTTCAGCTCGACCCCGTCC AGGCGGCATTTAACATTGGCGCGATGATCCGT TGGCTCGATTTCAACGATACCTGGCTGGCGGC GGAGTGGGGGCATCCTTCCGACAACCTCGGCG GCATTCTGGCAACGGCGGACTGGCTTTCGCGC AACGCGATCGCCAGCGGCAAAGCGCCGTTGAC CATGAAACAGGTGCTGACCGGAATGATCAAAG CCCATGAAATTCAGGGCTGCATCGCGCTGGAA AACTCCTTTAACCGCGTTGGTCTCGACCACGTT CTGTTAGTGAAAGTGGCTTCCACCGCCGTGGTC GCCGAAATGCTCGGCCTGACCCGCGAGGAAAT TCTCAACGCCGTTTCGCTGGCATGGGTAGACG GACAGTCGCTGCGCACTTATCGTCATGCACCG AACACCGGTACGCGTAAATCCTGGGCGGCGGG CGATGCTACATCCCGCGCGGTACGTCTGGCGC TGATGGCGAAAACGGGCGAAATGGGTTACCCG TCAGCCCTGACCGCGCCGGTGTGGGGTTTCTAC GACGTCTCCTTTAAAGGTGAGTCATTCCGCTTC CAGCGTCCGTACGGTTCCTACGTCATGGAAAA TGTGCTGTTCAAAATCTCCTTCCCGGCGGAGTT CCACTCCCAGACGGCAGTTGAAGCGGCGATGA CGCTCTATGAACAGATGCAGGCAGCAGGCAAA ACGGCGGCAGATATCGAAAAAGTGACCATTCG CACCCACGAAGCCTGTATTCGCATCATCGACA AAAAAGGGCCGCTCAATAACCCGGCAGACCGC	SEQ ID NO: 58

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	GACCACTGCATTCAGTACATGGTGGCGATCCC GCTGCTGTTCGGACGCTTAACGGCGGCAGATT ACGAGGACAACGTTGCGCAAGATAAACGCATC GACGCCCTGCGCGAGAAGATCAATTGCTTTGA AGATCCGGCGTTTACCGCTGACTACCACGACC CGGAAAAACGCGCCATCGCCAATGCCATAACC CTTGAGTTCACCGACGGCACACGATTTGAAGA AGTGGTGGTGGAGTACCCAATTGGTCATGCTC GCCGCCGTCAGGATGGCATTCCGAAGCTGGTC GATAAATTCAAAATCAATCTCGCGCGCCAGTT CCCGACTCGCCAGCAGCAGCGCATTCTGGAGG TTTCTCTCGACAGAACTCGCCTGGAACAGATG CCGGTCAATGAGTATCTCGACCTGTACGTCATT TAA
prpE sequence (comprised in the prpBCDE construct shown in FIG. 20)	ATGTCTTTTAGCGAATTTTATCAGCGTTCGATT AACGAACCGGAGAAGTTCTGGGCCGAGCAGGC CCGGCGTATTGACTGGCAGACGCCCTTTACGC AAACGCTCGACCACAGCAACCCGCCGTTTGCC CGTTGGTTTTGTGAAGGCCGAACCAACTTGTGT CACAACGCTATCGACCGCTGGCTGGAGAAACA GCCAGAGGCGCTGGCATTGATTGCCGTCTCTTC GGAAACAGAGGAAGAGCGTACCTTTACCTTCC GCCAGTTACATGACGAAGTGAATGCGGTGGCG TCAATGCTGCGCTCACTGGGCGTGCAGCGTGG CGATCGGGTGCTGGTGTATATGCCGATGATTG CCGAAGCGCATATTACCCTGCTGGCCTGCGCG CGCATTGGTGCTATTCACTCGGTGGTGTTTGGG GGATTTGCTTCGCACAGCGTGGCAACGCGAAT TGATGACGCTAAACCGGTGCTGATTGTCTCGG CTGATGCCGGGGCGCGCGGCGGTAAAATCATT CCGTATAAAAAATTGCTCGACGATGCGATAAG TCAGGCACAGCATCAGCCGCGTCACGTTTTACT GGTGGATCGCGGGCTGGCGAAAATGGCGCGCG TTAGCGGGCGGGATGTCGATTTCGCGTCGTTGC GCCATCAACACATCGGCGCGCGGGTGCCGGTG GCATGGCTGGAATCCAACGAAACCTCCTGCAT TCTCTACACCTCCGGCACGACCGGCAAACCTA AAGGTGTGCAGCGTGATGTCGGCGGATATGCG GTGGCGCTGGCGACCTCGATGGACACCATTTTT GGCGGCAAAGCGGGCGGCGTGTTCTTTTGTGC TTCGGATATCGGCTGGGTGGTAGGGCATTCGT ATATCGTTTACGCGCCGCTGCTGGCGGGGATG GCGACTATCGTTTACGAAGGATTGCCGACCTG GCCGGACTGCGGCGTGTGGTGGAAAATTGTCG AGAAATATCAGGTTAGCCGCATGTTCTCAGCG CCGACCGCCATTCGCGTGCTGAAAAAATTCCC TACCGCTGAAATTCGCAAACACGATCTTTCGTC GCTGGAAGTGCTCTATCTGGCTGGAGAACCGC	SEQ ID NO: 25

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TGGACGAGCCGACCGCCAGTTGGGTGAGCAAT ACGCTGGATGTGCCGGTCATCGACAACTACTG GCAGACCGAATCCGGCTGGCCGATTATGGCGA TTGCTCGCGGTCTGGATGACAGACCGACGCGT CTGGGAAGCCCCGGCGTGCCGATGTATGGCTA TAACGTGCAGTTGCTCAATGAAGTCACCGGCG AACCGTGTGGCGTCAATGAGAAAGGGATGCTG GTAGTGGAGGGGCCATTGCCGCCAGGCTGTAT TCAAACCATCTGGGGCGACGACGACCGCTTTG TGAAGACGTACTGGTCGCTGTTTTCCCGTCCGG TGTACGCCACTTTTGACTGGGGCATCCGCGATG CTGACGGTTATCACTTTATTCTCGGGCGCACTG ACGATGTGATTAACGTTGCCGGACATCGGCTG GGTACGCGTGAGATTGAAGAGAGTATCTCCAG TCATCCGGGCGTTGCCGAAGTGGCGGTGGTTG GGGTGAAAGATGCGCTGAAAGGGCAGGTGGC GGTGGCGTTTGTCATTCCGAAAGAGAGCGACA GTCTGGAAGACCGTGAGGTGGCGCACTCGCAA GAGAAGGCGATTATGGCGCTGGTGGACAGCCA GATTGGCAACTTTGGCCGCCCGGCGCACGTCT GGTTTGTCTCGCAATTGCCAAAAACGCGATCC GGAAAAATGCTGCGCCGCACGATCCAGGCGAT TTGCGAAGGACGCGATCCTGGGGATCTGACGA CCATTGATGATCCGGCGTCGTTGGATCAGATCC GCCAGGCGATGGAAGAGTAG

[0555] Next, the rate of propionate consumption of genetically engineered bacteria comprising the 2-Methylcitrate Cycle circuit is assessed in vitro.

[0556] Cultures of E. coli Nissle transformed with the plasmid comprising the prpBCDE circuit driven by the tet promoter and wild type control Nissle are grown overnight and then diluted 1:200 in LB. ATC is added to the cultures of the strain containing the prpEphaBCA construct plasmid at a concentration of 100 ng/mL to induce expression of the prpBCDE genes and the cells are grown with shaking at 250 rpm After 2 hrs of incubation, cells are pelleted down, washed, and resuspended in ImL M9 medium supplemented with glucose (0.5%) and propionate (8 mM) at a concentration of ~10⁹ cfu/ml bacteria. Aliquots are collected at 0 hrs, 2 hrs, and 4 hrs for propionate quantification and the catabolic rate is calculated.

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Example 15. Propionate Quantification in Bacterial Supernatant by LC-MS/MS

Sample Preparation [0557] Sodium propionate stock (10 mg/mL) in water was prepared, aliquoted in 1.5 mL microcentrifuge tubes (100 pL), and stored at -20°C. From the stock, Sodium propionate standards (1000, 500, 250, 100, 20, 4, 0.8 pg/mL) were prepared in water. Next, 25 pL of sample (bacterial supernatant and standards) was mixed with 75 pL of ACN/H2O (45:30, v/v) containing lOpg/mL of sodium propionate-d5 in a round-bottom 96-well plate. The plates were heat sealed with a PierceASeal foil and mixed well.

[0558] In a V-bottom 96-well polypropylene plate, 5 pL of diluted samples were added to 95 pL of derivatization mix (20mM EDC [A-(3-Dimethylaminopropyl)-A'ethylcarbodiimide hydrochloride] and 20mM TFEA [2,2,2-Trifluoroethylamine hydrochloride] in 10 mM MES buffer pH 4.0). The plates were heat sealed with a ThermASeal foil and mixed well. The samples were incubated at RT for lhr for derivatization and then centrifuged at 4000rpm for 5min.

[0559] Next, 20 pL of the solution were transferred into a round-bottom 96-well plate, and 180 uL 0.1% formic acid in water was added to the samples. The plates were heatsealed and mixed as described above.

LC-MS/MS method [0560] Derivatized propionate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Method details are described in Table 17 and Table

18. Tandem Mass Spectrometry details are described in Table 19.

Table 17. HPLC Method Details

Column	Aquasil Cl8(2) column, 5 pm (50 x 2.1 mm)
Mobile Phase A	100% H2O, 0.1% Formic Acid
Mobile Phase B	100% ACN, 0.1% Formic Acid
Injection volume	lOuL

Table 18. HPLC Method Details

Time (min)	Flow Rate	A%	B%
-0.5	250	100	0

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0.5	250	100	0
2.5	250	10	90
3.5	250	10	90
3.51	250	0	10

Table 19. Tandem Mass Spectrometry Details

Ion Source	HESI-II
Polarity	Positive
SRM transitions
Sodium propionate	156.2/57.1
Sodium propionate-d5	161.0/62.1

Example 16. Acetylcamitine and Propionylcarnitine Quantification in Plasma and Urine by LC-MS/MS

Sample Preparation [0561] Acetylcamitine and Propionylcarnitine stock (10 mg/mL) was prepared in water, aliquoted into 1.5 mL microcentrifuge tubes (100 pL), and stored at -20°C. Standards of 250, 100, 20, 4, 0.8, 0.16, 0.032 pg/mL were prepared in water. Sample (10 pL) and standards were mixed with 90 pL of ACN/MeOH/fUO (60:20:10, v/v) containing 1 pg/mL of Acetylcarnitine-d3 and Propionylcarnitine-d3 in the final solution) in a V-bottom 96-well plate. The plate was heat sealed with a AlumASeal foil, mixed well, and centrifuged at 4000rpm for 5min. Next, 20pL of the solution was transferred into a round-bottom 96-well plate, and 180 uL 0.1% formic acid in water was added to the sample. The plate was heatsealed with a ClearASeal sheet and mixed well.

LC-MS/MS method [0562] Propionylcarnitine and Acetylcamitine were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Method details are described in Table 20 and Table 21. Tandem Mass Spectrometry details are described in Table 22.

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Table 20. HPLC Method Details

Column	HILIC column, 2.6 pm (100 x 2.1 mm)
Mobile Phase A	100% H2O, 0.1% Formic Acid
Mobile Phase B	100% ACN, 0.1% Formic Acid
Injection volume	lOuL

Table 21. HPLC Method Details

Time (min)	Flow Rate (pL/min)	A%	B%
-0.5	250	100	0
0.5	250	100	0
2.5	250	10	90
3.5	250	10	90
3.51	250	0	10

[03]

Table 22. Tandem Mass Spectrometry Details

Ion Source	HESI-II
Polarity	Positive
SRM transitions
Acetylcarnitine	204.1/85.2
Acetylcarnitine-d3	207.1/85.2
Propionylcarnitine	218.1/85.2
Propionylcarnitine-d3	221.1/85.2

Example 17. Propionate, 2-Methylcitrate, Propionylglycine, and Tigloylglycine

Quantification in Plasma and Urine by UC-MS/MS

Sample Preparation [0563] Stocks of 10 mg/mL Sodium propionate, 2-Methylcitrate, Propionylglycine, and Tigloylglycine in water were prepared, aliquoted in 1.5 mL microcentrifuge tubes (100 pL), and stored at -20°C. Standards of 500, 250, 100, 20, 4, 0.8, 0.16, 0.032 pg/mL of each of the stocks were prepared in water. On ice, 10pL of sample (and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90pL of the derivatizing solution containing 50mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphospine in acetonitrile with 5 ug/mL of Sodium propionate-13C3 and 2-Methylcitrate-d3 were added into the final solution. The plate was heat sealed with a ThermASeal foil and mixed well. The

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PCT/US2016/044922 samples were incubated at 60°C for lhr for derivatization and then centrifuged at 4000rpm for 5min. Next, 20pL of the derivatized samples were added to 180pL of 0.1% formic acid in water/ACN (140:40) in a round-bottom 96-well plate. The plate was heat sealed with a Clear AS eal sheet and mix well.

LC-MS/MS method [0564] Derivatized metabolites were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Method details are described in Table 23 and Table

24. Tandem Mass Spectrometry details are described in Table 25.

Table 23. HPLC Method Details

Column	C18 column, 5 gm (100 x 2 mm)
Mobile Phase A	100% H2O, 0.1% Formic Acid
Mobile Phase B	100% ACN, 0.1% Formic Acid
Injection volume	lOuL

Table 24. HPLC Method Details

Time (min)	Flow Rate (gL/min)	A%	B%
0	500	95	5
0.9	500	95	5
1.0	500	72.5	27.5
2.5	500	60	40
2.6	500	10	90
4.5	500	10	90
4.51	500	95	5
4.75	500	95	5

Table 25. Tandem Mass Spectrometry Details

Ion Source	HESI-II
Polarity	Positive
SRM transitions
Sodium propionate	216.1/160.1
Sodium propionate-13C3	219.1/160.1
2-Methylcitrate	489.2/471.2
2-Methylcitrate-d3	492.2/474.2
Propionylqlycine	273.1/172.2*

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Tigloylglycine| 299.1/160.1 * *Quantified using external calibration (without internal standard)

Example 18. In Vivo Studies Demonstrating that the Engineered Bacterial Cells

Decrease Propionate Concentration [0565] For in vivo studies, a hypomorphic mouse model of propionic acidemia is used (see, for example Guenzel et al., 2013). Alternatively, a PCCA-/- knock-out mouse or a mouse model of methylmalonic acidemia can be used (see, for example, Miyazaki et al.,

2001 or Peters et al., 2012). Briefly, blood levels of methylcitrate, acetylcarnitine, and/or propionylcarnitine are measured in the mice prior to administration of the engineered bacteria on day 0. On day 1, cultures of E. coli Nissle containing pTet-prpBCDE and/or pTet-mctC are administered to three wild-type mice and three hypomorph mice once daily for a week.

In addition, three hypomorph mice are administered PBS as a control once daily for a week. Treatment efficacy is determined, for example, by measuring blood levels of methylcitrate, acetylcarnitine, and/or propionylcarnitine. A decrease in blood levels of methylcitrate, acetylcarnitine, and/or propionylcarnitine after treatment with the engineered bacterial cells indicates that the engineered bacterial cells are effective for treating propionic acidemia and methylmalonic acidemia. Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with PA or MMA, for example, seizures, further indicates the efficacy of the engineered bacterial cells for treating PA and MMA.

Example 19. Generation of AThyA [0566] An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thy A, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.

[0567] A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows.

Sequences of the primers used at a lOOum concentration are found in Table 26.

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Table 26. Primer Sequences

Nam e	Sequence	Description	SEQ ID NO
SR36	tagaactgatgcaaaaagtgctcgacgaaggcacacagaTGTGTAGG CTGGAGCTGCTTC	Round 1: binds on pKD3	SEQ ID NO: 59
SR38	gtttcgtaattagatagccaccggcgctttaatgcccggaCATATGAAT ATCCTCCTTAG	Round 1: binds on pKD3	SEQ ID NO: 60
SR33	caacacgtttcctgaggaaccatgaaacagtatttagaactgatgcaaaaag	Round 2: binds to round 1 PCR product	SEQ ID NO: 61
SR34	cgcacactggcgtcggctctggcaggatgtttcgtaattagatagc	Round 2: binds to round 1 PCR product	SEQ ID NO: 62
SR43	atatcgtcgcagcccacagcaacacgtttcctgagg	Round 3: binds to round 2 PCR product	SEQ ID NO: 63
SR44	aagaatttaacggagggcaaaaaaaaccgacgcacactggcgtcggc	Round 3: binds to round 2 PCR product	SEQ ID NO: 64

[0568] For the first PCR round, 4x50ul PCR reactions containing Ing pKD3 as template, 25ul 2xphusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions:

[0569] stepl: 98c for 30s [0570] step2: 98c for 10s [0571] step3: 55c for 15s [0572] step4: 72c for 20s [0573] repeat step 2-4 for 30 cycles [0574] step5: 72c for 5min [0575] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer’s instructions and eluted in 30ul nuclease free water.

[0576] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction.

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The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[0577] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul FB containing 3mM thymidine and spread on FB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on FB plates were restreaked. + cam 20ug/ml + or - thy 3mM. (thyA auxotrophs will only grow in media supplemented with thy 3mM).

[0578] Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FFP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in FB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. lmF of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in lmF ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmF SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for lhours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul FB containing 3mM thymidine and spread on FB agar plates containing 3mM thy and lOOug/ml carbenicillin and grown at 30°C for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42°C.

[0579] To test the colony-purified transformants, a colony was picked from the 42°C plate with a pipette tip and resuspended in 10pF FB. 3pF of the cell suspension was pipetted

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PCT/US2016/044922 onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.

[0580] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer’s instructions and eluted in 30ul nuclease free water.

[0581] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[0582] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. (thyA auxotrophs will only grow in media supplemented with thy 3mM).

[0583] Next, the antibiotic resistance was removed with pCP20 transformation.

pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the

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PCT/US2016/044922 selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. lmL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in lmL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for lhours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and lOOug/ml carbenicillin and grown at 30°C for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42°C.

[0584] To test the colony-purified transformants, a colony was picked from the 42°C plate with a pipette tip and resuspended in 10pL LB. 3pL of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.

Example 20. Nitric oxide-inducible reporter constructs [0585] ATC and nitric oxide-inducible reporter constructs were synthesized (Genewiz, Cambridge, MA). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrRGFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations (FIG. 43); promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and

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

[0586] FIG. 43D shows a dot blot of NO-GFP constructs. E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1:100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS-treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

[0587] Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrRinducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1:100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered

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PCT/US2016/044922 from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. FIG. 43 shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

Example 21. FNR promoter activity [0588] In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 3. The nucleotide sequences of these constructs are shown in Tables 27-31 (SEQ ID NO: 65-69). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity, exemplary results are shown in FIG. 41.

[0589] Table 27 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnrl (SEQ ID NO: 65). The construct comprises a translational fusion of the Nissle nirB 1 gene and the lacZ gene, in which the translational fusions are fused in frame to the 8th codon of the lacZ coding region. The Pfnrl sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0590] Table 28 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr2 (SEQ ID NO: 66). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8th codon of the lacZ coding region. The Pfnr2 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

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PCT/US2016/044922 [0591] Table 29 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr3 (SEQ ID NO: 67). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The Pfnr3 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0592] Table 30 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr4 (SEQ ID NO: 68). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The Pfnr4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0593] Table 31 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS (SEQ ID NO:

69). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrSl, fused to lacZ. The Pfnrs sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 27. Pfnrl-lacZ Construct Sequences

Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 65)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtc gtccggccttttcctctcttactctgctacgtacatctatttctataaatccgttcaatttg tctgttttttgcacaaacatgaaatatcagacaattccgtgacttaagaaaatttatacaaa tcagcaatataccccttaaggagtatataaaggtgaatttgatttacatcaataagcggggt tgctgaatcgttaaggtaggcggtaatagaaaagaaatcgaggcaaaaATGagcaaagtcag act cgcaattatGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCG TTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAG GCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTG GTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATA CTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTG ACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTC

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Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 65)

GCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATG

GCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGC

CGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGT

GATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCG

GCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTT

ACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGG

CGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCA

GCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTC

ACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCG

TGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCG

GTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATT

CGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGAT

GGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATC

CGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCC

AATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACC

CGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCA

TCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATC

AAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCAC

CGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGA

AATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAA

TATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCA

GTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATG

ATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGC

CAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGC

AAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCG

AATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAG

CCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACT

GCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAAC

CAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCG

GAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGG

AACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTC

TTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACC

CGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTG

GGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGG

CAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAA

ACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGT

GGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGG

CGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGC

CTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGT

CTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGT

GGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGC

CATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGG

GATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTC

GCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

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Table 28. Pfnr2-lacZ Construct Sequences

Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 66)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatg catgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacaggagtat ttatattgcgcccgttacgtgggcttcgactgtaaatcagaaaggagaaaacacctATGacg acctacgatcgGGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGT TACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG

CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGG

TTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATAC

TGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGA

CCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCG

CTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGG

CGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCC

GTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTG

ATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGG

CATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTA

CCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGC

GAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAG

CGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCA

CACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGT

GCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGG

TTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTC

GCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATG

GTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCC

GAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCA

ATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCC

GCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCAT

CTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCA

AATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACC

GATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAA

ATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAAT

ATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAG

TACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGA

TGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCC

AGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCA

AAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGA

ATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGC

CGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTG

CCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACC

AAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGG

AAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGA

ACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCT

TTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCC

GTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGG

GTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGC

AGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAA

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Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 66)

CCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTG

GATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGC

GCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCC

TTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTC

TTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTG

GCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCC

ATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGG

ATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCG

CTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 29. Pfnr3-lacZ Construct Sequences

Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 67)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtc gtccggccttttcctctcttactctgctacgtacatctatttctataaatccgttcaatttg tctgttttttgcacaaacatgaaatatcagacaattccgtgacttaagaaaatttatacaaa tcagcaatataccccttaaggagtatataaaggtgaatttgatttacatcaataagcggggt tgctgaatcgttaaGGATCCctctagaaataattttgtttaactttaagaaggagatataca tATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA

GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGC

CTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCG

ATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAAC

GTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTA

CTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTG

ATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGAC

AGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGC

GGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGA

GCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAA

GTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTA

CGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCG

CCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGC

GTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTA

TCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACG

TCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTG

ATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGAC

GATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATT

ATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAA

GCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCT

ACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGA

TCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGG

ATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGC

CACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGC

CGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGC

-283WO 2017/023818

PCT/US2016/044922

Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 67)

GAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCG

TCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAAT

ATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGAT

CGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGA

AGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCA

GCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGC

AAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGA

ACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGC

AACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTG

GCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAG

CGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCT

TTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTC

ACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGC

CTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCA

CGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGG

AAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAA

TGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGC

TGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGAC

CGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTA

CGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACC

AGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACC

AGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATAT

GGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCG

GTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 30. Pfnr4-lacZ construct Sequences

Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 68)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatg catgcatcaaaaaagatgtgagcttgatcaaaaacaaaaaatatttcactcgacaggagtat ttatattgcgcccGGATCCctctagaaataattttgtttaactttaagaaggagatatacat ATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGG CGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAG

AGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCC

TGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGA

TACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACG

TGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTAC

TCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGA

TGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACA

GCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCG

GTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAG

CGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAG

TTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTAC

GGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGC

-284WO 2017/023818

PCT/US2016/044922

Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 68)

CAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCG

TCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTAT

CGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGT

CGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGA

TTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACG

ATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTA

TCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAG

CCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTA

CCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGAT

CATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGA

TCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCC

ACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCC

GAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCG

AATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGT

CAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATA

TGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATC

GCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAA

GCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAG

CGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCA

AGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAA

CTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCA

ACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGG

CGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGC

GGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTT

TCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCA

CCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCC

TGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCAC

GGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGA

AAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAAT

GTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCT

GGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACC

GCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTAC

GTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCA

GTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCA

GCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATG

GGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGG

TCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 31. Pfnrs-lacZ construct Sequences

Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 69)

GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgtaac aaaagcaatttttccggctgtctgtatacaaaaacgccgtaaagtttgagcgaagtcaataa actctctacccattcagggcaatatctctcttGGATCCctctagaaataattttgtttaact

-285WO 2017/023818

PCT/US2016/044922

Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 69)

ttaagaaggagatatacatATGCTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCG

TGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCA

GCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT

GGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTG

CGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATG

CGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAG

AATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCA

GACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGG

TCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCC

GGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCA

GGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGC

AAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAG

GCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCA

GGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTG

GCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCC

GAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGA

AGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGA

ACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAG

GTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGC

CGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCC

TGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACC

GATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCG

TAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACG

ACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGC

GGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCA

GCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGC

GCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAA

TACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGA

TCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTG

GCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCG

CATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCG

AACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGA

TGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAA

GGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCT

AACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCT

GGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATC

CCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATT

TAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCC

CGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACC

CGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGC

GGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACG

CGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCAC

GGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGAT

TGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGC

AAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCA

GACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATT

GAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAAC

AACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAAT

-286WO 2017/023818

PCT/US2016/044922

Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 69)

ATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGA

ATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Example 22. Sequences [0594] In some embodiments, the genetically engineered bacteria comprise a gene cassette which is driven by a propionate responsive promoter. In a non-limiting example, the gene cassette is driven by the prpR Propionate-Responsive promoter. In a non-limiting example, the prpR Propionate-Responsive promoter has the sequence shown in Table 32.

Table 32. prpR Propionate-Responsive Promoter Sequence

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PCT/US2016/044922 gaacgggcatagagtgtaatcgtatggcgaa

CCTGCTCCATTTGTGGTGAATCGCCGAG atc^tca^^at^cgggtctgtaatccc

TTGCTGGAGGTATGCTGGCTATACTGACGCC gtgtcaggcgggtcatatcc^^ gaaagcctgacgtacggtggccgct

ATAAAGATGGCGGTCATTCCTG^ caggtcggtaattagtcctgX gcctcaatgccgttagctttgagct

TTGCCCGCGAGCATCCTCTTCAGTGATATAGC ttcgctgttcaagacggaggtgaaacgtttt

CTGAAAGGCGACCAGAGCCGGAATGGTCTC'c

TGATAGGTCACGATTCCCATTC

GCTTTCCCGCTTTTGCCAGAGCCT

TCGAATCCGCTGGGTTTGATGAGG^

GTACCGACAGTC gttggaacctgXgcgataatX

CGTTCGGTTGCCAGTTTTTTGCGAA

TACTGCCTTTTCAAAACCGAGCTGA

GTGATCGTCGCCAGATGATCM

TGATATCCCGAAATAGTTCGA

TACCGAGACCGTCCAGATCA

CTATTATCGCGCGAAGCGCTATGCACAGTAA

CCATCGTCGTAGATTCATGTTTAAGGAACGA

ATTCTTGTTTTATAGATGTTTCGTTAATGTTG

CAATGAAACACAGGCCTCCGTTTCATGAAAC

GTTAGCTGACTCGTTTTTCTTGTGACTCGTCT

GTCAGTATTAAAAAAGATTTTTCATTTAACTG

ATTGTTTTTAAATTGAATTTTATTTAATGGTT

TCTCGGTTTTTGGGTCTGGCATATCCCTTGCT

TTAATGAGTGCATCTTAATTAACAATTCAATA

ACAAGAGGGCTGAATagtaatttcaacaaaataacgagcatt cgaatg

-288WO 2017/023818

PCT/US2016/044922

Table 33. List of Sequences

SEQ ID NO:	Gene or Gene Cassette	Origin	Sequence
71	PrpE	E. coli	MSFSEFYQRSINEPEKFWAEQARRIDWQTPFTQTLDHSNPP FARWFCEGRTNLCHNAIDRWLEKQPEALALIAVSSETEEER TFTFRQLHDEVNAVASMLRSLGVQRGDRVLVYMPMIAEA HITLLACARIGAIHSVVFGGFASHSVATRIDDAKPVLIVSAD AGARGGKIIPYKKLLDDAISQAQHQPRHVLLVDRGLAKMA RVSGRDVDFASLRHQHIGARVPVAWLESNETSCILYTSGTT GKPKGVQRDVGGYAVALATSMDTIFGGKAGGVFFCASDI GWVVGHSYIVYAPLLAGMATIVYEGLPTWPDCGVWWKIV EKYQVSRMFSAPTAIRVLKKFPTAEIRKHDLSSLEVLYLAG EPLDEPTASWVSNTLDVPVIDNYWQTESGWPIMAIARGLD DRPTRLGSPGVPMYGYNVQLLNEVTGEPCGVNEKGMLVV EGPLPPGCIQTIWGDDDRFVKTYWSLFSRPVYATFDWGIR DADGYHFILGRTDDVINVAGHRLGTREIEESISSHPGVAEV AVVGVKDALKGQVAVAFVIPKESDSLEDREVAHSQEKAI MALVDSQIGNFGRPAHVWFVSQLPKTRSGKMLRRTIQAIC EGRDPGDLTTIDDPASLDQIRQAMEE
72	PrpE	Salmonella	MSFSEFYQRSINEPEQFWAEQARRIDWQQPFTQTLDYSNPP FARWFCGGTTNLCHNAIDRWLDTQPDALALIAVSSETEEE RTFTFRQLYDEVNVVASMLLSLGVRRGDRVLVYMPMIAE AHITLLACARIGAIHSVVFGGFASHSVAARIDDARPVLIVSA DAGARGGKVIPYKKLLDEAVDQAQHQPKHVLLVDRGLAK MARVAGRDVDFATLREHHAGARVPVAWLESNESSCILYT SGTTGKPKGVQRDVGGYAVALATSMDTLFGGKAGGVFFC ASDIGWVVGHSYIVYAPLLAGMATIVYEGLPTYPDCGVW WKIVEKYRVSRMFSAPTAIRVLKKFPTAQIRNHDLSSLEVL YLAGEPLDEPTAAWVSGTLGVPVIDNYWQTESGWPIMAL ARTLDDRPSRLGSPGVPMYGYNVQLLNEVTGEPCGANEK GMVVIEGPLPPGCIQTIWGDDARFVNTYWSLFTRQVYATF DWGIRDADGYYFILGRTDDVINVAGHRLGTREIEESISSYP NVAEVAVVGVKDALKGQVAVAFVIPKQSDSLEDREVAHS EEKAIMALVDSQIGNFGRPAHVWFVSQLPKTRSGKMLRRT IQAICEGRDPGDLTTIDDPTSLQQIRQVIEE
73	prpE	Salmonella	ATGTCTTTTAGCGAATTTTATCAGCGTTCGATTAACGAA CCGGAGCAGTTCTGGGCTGAACAGGCCCGGCGTATCGA CTGGCAGCAGCCGTTTACGCAGACGCTGGACTACAGCA ACCCGCCGTTTGCCCGCTGGTTTTGCGGCGGCACCACTA ATCTGTGCCATAACGCGATTGACCGCTGGCTGGATACCC AGCCGGATGCGCTGGCGCTGATTGCGGTTTCCTCTGAGA CCGAAGAAGAACGTACCTTCACCTTTCGTCAACTGTATG ACGAGGTGAATGTCGTGGCCTCTATGCTGCTGTCACTGG GCGTGCGGCGTGGCGATCGGGTACTGGTGTATATGCCG ATGATTGCCGAGGCGCACATCACATTACTGGCCTGCGCG CGCATTGGCGCGATCCATTCAGTGGTGTTTGGTGGTTTT GCCTCGCACAGTGTAGCCGCGCGCATCGACGATGCCAG ACCGGTGCTGATTGTCTCGGCGGACGCCGGAGCGCGAG GTGGGAAGGTCATTCCCTATAAAAAGCTTCTTGATGAGG CGGTCGATCAGGCACAGCATCAGCCGAAGCATGTACTG CTGGTGGATCGGGGGCTGGCGAAAATGGCGCGGGTTGC

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PCT/US2016/044922

			CGGGCGCGATGTGGATTTTGCGACCCTGCGCGAACACC ATGCCGGGGCGCGTGTGCCAGTGGCCTGGCTTGAATCTA ATGAAAGTTCCTGCATTCTTTATACCTCCGGCACTACCG GCAAACCGAAAGGCGTTCAGCGTGACGTTGGTGGCTAC GCCGTGGCGCTGGCGACATCGATGGACACCCTCTTTGGC GGCAAAGCGGGCGGCGTCTTTTTCTGCGCTTCGGATATC GGTTGGGTAGTGGGGCACTCTTATATTGTGTATGCGCCG CTGCTGGCGGGTATGGCGACCATCGTTTATGAAGGATTG CCGACGTATCCGGACTGCGGCGTATGGTGGAAAATTGTC GAGAAATATCGGGTGAGCCGGATGTTTTCAGCGCCAAC CGCCATTCGTGTGCTGAAGAAATTTCCCACCGCGCAGAT ACGCAATCATGATCTCTCCTCGCTGGAAGTTCTCTATCT GGCAGGCGAGCCGCTCGACGAGCCAACGGCAGCCTGGG TTAGCGGAACACTGGGTGTGCCGGTGATCGACAATTACT GGCAGACCGAATCCGGCTGGCCGATTATGGCGCTGGCG CGCACGCTTGATGACAGACCATCGCGTTTGGGCAGTCCC GGCGTGCCGATGTACGGCTATAATGTTCAACTGCTCAAC GAGGTGACCGGTGAACCCTGTGGTGCGAACGAAAAGGG AATGGTGGTTATTGAAGGGCCGCTGCCGCCGGGCTGCAT TCAGACCATCTGGGGCGATGACGCACGCTTTGTGAATAC CTACTGGTCACTGTTTACTCGTCAGGTGTATGCCACCTTT GACTGGGGGATCCGCGACGCCGACGGCTATTATTTTATC CTTGGGCGCACGGATGATGTGATCAACGTCGCCGGACA TCGTCTCGGCACCCGTGAGATAGAGGAGAGCATCTCCA GCTATCCCAACGTTGCGGAAGTGGCGGTGGTAGGGGTA AAAGACGCGCTGAAAGGGCAGGTAGCGGTAGCCTTCGT GATCCCGAAACAGAGTGACAGTCTGGAAGACCGCGAAG TGGCGCATTCGGAAGAGAAGGCGATTATGGCGCTGGTC GATAGTCAGATCGGCAACTTTGGCCGCCCGGCGCACGT GTGGTTTGTCTCGCAGCTACCAAAAACCCGATCCGGGAA GATGCTCAGACGAACGATCCAGGCGATCTGCGAGGGCC GGGATCCAGGCGATCTGACGACCATTGACGATCCGACG TCGTTGCAACAAATTCGCCAGGTCATTGAGGAGTAA
74	PrpC	E. coli	MSDTTILQNSTHVIKPKKSVALSGVPAGNTALCTVGKSGN DLHYRGYDILDLAEHCEFEEVAHLLIHGKLPTRDELAAYK TKLKALRGLPANVRTVLEALPAASHPMDVMRTGVSALGC TLPEKEGHTVSGARDIADKLLASLSSILLYWYHYSHNGERI QPETDDDSIGGHFLHLLHGEKPSQSWEKAMHISLVLYAEH EFNASTFTSRVIAGTGSDMYSAIIGAIGALRGPKHGGANEV SLEIQQRYETPGEAEADIRKRVENKEVVIGFGHPVYTIADP RHQVIKRVAKQLSQEGGSLKMYNIADRLETVMWESKKMF PNLDWFSAVSYNMMGVPTEMFTPLFVIARVTGWAAHIIEQ RQDNKIIRPSANYVGPEDRQFVALDKRQ
75	PrpC	Salmonella	MSDTTILQNNTNVIKPKKSVALSGVPAGNTALCTVGKSGN DLHYRGYDILDLAEHCEFEEVAHLLIHGKLPTRDELNAYK SKLKALRGLPANVRTVLEALPAASHPMDVMRTGVSALGC TLPEKEGHTVSGARDIADKLLASLSSILLYWYHYSHNGERI QPETDDDSIGGHFLHLLHGEKPSQSWEKAMHISLVLYAEH EFNASTFTSRVVAGTGSDMYSAIIGAIGALRGPKHGGANEV SLEIQQRYETPDEAEADIRKRIANKEVVIGFGHPVYTIADPR HQVIKRVAKQLSQEGGSLKMYNIADRLETVMWDSKKMFP NLDWFSAVSYNMMGVPTEMFTPLFVIARVTGWAAHIIEQR QDNKIIRPSANYIGPEDRAFTPLEQRQ
76	prpC	Salmonella	ATGAGCGACACGACGATCCTGCAAAACAACACAAATGT CATTAAGCCAAAAAAATCCGTCGCATTATCCGGCGTACC CGCCGGAAATACCGCCTTATGCACCGTAGGTAAAAGCG GTAACGATCTGCACTATCGCGGGTACGATATTCTCGATC TCGCGGAGCACTGTGAATTTGAAGAAGTTGCGCATCTGC

-290WO 2017/023818

PCT/US2016/044922

			TCATTCACGGCAAGCTGCCCACCCGTGATGAGCTGAATG CCTATAAAAGCAAATTAAAAGCGCTGCGTGGCTTACCC GCTAACGTCCGTACCGTGCTGGAAGCGCTGCCAGCGGC ATCGCACCCGATGGACGTAATGCGCACCGGCGTTTCTGC GCTGGGCTGCACCCTGCCGGAAAAAGAGGGGCATACCG TTTCTGGCGCGCGTGATATCGCCGACAAGCTGCTGGCCT CCCTCAGCTCCATTCTCCTTTACTGGTATCACTACAGCCA CAACGGCGAACGCATTCAGCCAGAAACTGACGATGACT CTATCGGCGGGCATTTCCTGCATTTATTACACGGCGAAA AGCCATCGCAAAGCTGGGAAAAGGCGATGCACATTTCA CTGGTACTGTACGCCGAACATGAGTTCAACGCCTCAACC TTTACCAGCCGGGTGGTAGCCGGTACGGGATCGGATAT GTACTCCGCCATCATTGGCGCGATAGGCGCGCTTCGCGG GCCGAAGCACGGCGGGGCGAATGAAGTCTCGCTGGAGA TTCAGCAGCGCTACGAAACGCCGGATGAAGCAGAAGCC GATATCCGTAAACGTATCGCCAATAAAGAAGTGGTGAT TGGTTTTGGTCATCCGGTATACACCATCGCCGATCCGCG CCATCAGGTGATTAAGCGGGTAGCGAAGCAGCTTTCAC AGGAGGGCGGTTCGCTGAAGATGTACAACATTGCCGAT CGGCTGGAGACGGTAATGTGGGACAGCAAAAAGATGTT CCCTAATCTCGACTGGTTCTCGGCGGTCTCCTACAACAT GATGGGCGTTCCCACCGAAATGTTTACCCCGCTGTTTGT GATTGCCCGCGTTACAGGTTGGGCGGCGCACATCATCGA GCAACGACAGGACAACAAAATTATCCGTCCTTCCGCCA ATTATATTGGCCCGGAAGATCGCGCCTTTACGCCGCTGG AACAGCGTCAGTAA
77	PrpD	E. coli	MSAQINNIRPEFDREIVDIVDYVMNYEISSRVAYDTAHYCL LDTLGCGLEALEYPACKKLLGPIVPGTVVPNGVRVPGTQF QLDPVQAAFNIGAMIRWLDFNDTWLAAEWGHPSDNLGGI LATADWLSRNAIASGKAPLTMKQVLTGMIKAHEIQGCIAL ENSFNRVGLDHVLLVKVASTAVVAEMLGLTREEILNAVSL AWVDGQSLRTYRHAPNTGTRKSWAAGDATSRAVRLALM AKTGEMGYPSALTAPVWGFYDVSFKGESFRFQRPYGSYV MENVLFKISFPAEFHSQTAVEAAMTLYEQMQAAGKTAADI EKVTIRTHEACIRIIDKKGPLNNPADRDHCIQYMVAIPLLFG RLTAADYEDNVAQDKRIDALREKINCFEDPAFTADYHDPE KRAIANAITLEFTDGTRFEEVVVEYPIGHARRRQDGIPKLV DKFKINLARQFPTRQQQRILEVSLDRTRLEQMPVNEYLDLY VI
78	PrpD	Salmonella	MSAPVSNVRPEFDREIVDIVDYVMKYNITSKVAYDTAHYC LLDTLGCGLEALEYPACKKLMGPIVPGTVVPNGVRVPGTQ FQLDPVQAAFNIGAMIRWLDFNDTWLAAEWGHPSDNLGG ILATADWLSRNAVAAGKAPLTMQQVLTGMIKAHEIQGCIA LENSFNRVGLDHVLLVKVASTAVVAEMLGLTRDEILNAVS LAWVDGQSLRTYRHAPNTGTRKSWAAGDATSRAVRLAL MAKTGEMGYPSALTAKTWGFYDVSFKGEKFRFQRPYGSY VMENVLFKISFPAEFHSQTAVEAAMTLYEQMQAAGKTAA DIEKVTIRTHEACIRIIDKKGPLNNPADRDHCIQYMVAIPLL FGRLTAADYEDGVAQDKRIDALREKTHCFEDPAFTTDYHD PEKRSIANAISLEFTDGTRFDEVVVEYPIGHARRRGDGIPKL IEKFKINLARQFPPRQQQRILDVSLDRTRLEQMPVNEYLDL YVI
79	prpD	Salmonella	ATGTCCGCACCTGTTTCGAACGTCCGCCCTGAATTTGAC CGTGAAATTGTTGATATTGTTGATTATGTGATGAAGTAC AACATCACCTCAAAAGTGGCTTATGACACCGCGCACTAC TGTCTGCTTGATACCCTGGGCTGTGGGCTGGAAGCGCTG GAATATCCGGCCTGTAAAAAATTGATGGGGCCTATCGTG CCAGGTACCGTGGTGCCGAACGGTGTACGTGTACCGGG

-291WO 2017/023818

PCT/US2016/044922

			CACTCAGTTCCAGCTCGATCCGGTGCAGGCGGCATTTAA TATTGGCGCGATGATCCGCTGGCTCGACTTTAACGATAC CTGGCTTGCCGCTGAGTGGGGACACCCTTCCGATAACCT CGGCGGTATTCTGGCGACCGCCGACTGGTTGTCGCGCAA CGCCGTCGCCGCCGGTAAAGCGCCGCTGACCATGCAGC AGGTGCTGACCGGGATGATCAAAGCCCACGAAATCCAG GGCTGTATCGCGCTGGAAAACTCGTTTAACCGCGTGGGT CTCGATCACGTTTTGCTGGTGAAAGTGGCTTCCACGGCT GTAGTGGCTGAAATGCTCGGCCTGACCCGCGATGAAATT CTCAACGCCGTATCGCTGGCGTGGGTGGATGGGCAGTC GCTGCGTACCTATCGCCATGCGCCAAACACCGGTACGCG CAAATCCTGGGCGGCAGGCGATGCCACTTCACGCGCGG TGCGTCTGGCGCTGATGGCGAAAACTGGCGAGATGGGC TATCCCTCGGCGTTGACCGCCAAAACCTGGGGCTTTTAT GACGTCTCGTTCAAAGGCGAAAAATTCCGTTTCCAGCGC CCGTACGGCTCCTACGTGATGGAAAACGTGCTGTTCAAA ATCTCCTTCCCGGCGGAGTTCCATTCGCAGACCGCCGTT GAAGCAGCGATGACGCTGTATGAGCAGATGCAGGCGGC TGGAAAAACGGCGGCGGATATCGAAAAAGTAACGATTC GCACCCATGAAGCCTGTATACGCATCATTGATAAAAAA GGCCCGCTGAATAATCCGGCTGACCGCGATCACTGTATT CAGTATATGGTGGCGATCCCACTGCTGTTCGGACGCTTA ACGGCGGCGGATTATGAGGATGGCGTGGCGCAGGATAA ACGTATTGACGCGCTGCGTGAAAAAACGCATTGCTTTGA AGACCCGGCGTTTACCACTGATTATCATGACCCGGAAAA ACGTTCGATTGCCAACGCCATTAGTCTTGAATTTACTGA CGGTACCCGTTTTGACGAGGTGGTTGTCGAGTACCCGAT CGGCCACGCGCGTCGTCGCGGCGACGGCATTCCAAAAC TTATCGAAAAATTTAAAATCAATCTGGCGCGCCAGTTCC CACCCCGCCAGCAACAACGCATCCTGGATGTCTCCCTGG ACAGAACGCGCCTGGAGCAGATGCCGGTTAATGAGTAT CTCGACTTGTACGTCATCTAG
80	PrpB	E. coli	MSLHSPGKAFRAALSKETPLQIVGTINANHALLAQRAGYQ AIYLSGGGVAAGSLGLPDLGISTLDDVLTDIRRITDVCSLPL LVDADIGFGSSAFNVARTVKSMIKAGAAGLHIEDQVGAKR CGHRPNKAIVSKEEMVDRIRAAVDAKTDPDFVIMARTDAL AVEGLDAAIERAQAYVEAGAEMLFPEAITELAMYRQFAD AVQVPILSNITEFGATPLFTTDELRSAHVAMALYPLSAFRA MNRAAEHVYNILRQEGTQKSVIDTMQTRNELYESINYYQY EEKLDDLFARGQVK
81	PrpB	Salmonella	MTLHSPGQAFRAALAKEKPLQIVGAINANHALLAQRAGY QALYLSGGGVAAGSLGLPDLGISTLDDVLTDIRRITDVCPL PLLVDADIGFGSSAFNVARTVKSISKAGAAALHIEDQIGAK RCGHRPNKAIVSKEEMVDRIHAAVDARTDPDFVIMARTDA LAVEGLDAAIDRARAYVEAGADMLFPEAITELAMYRQFA DAVQVPILANITEFGATPLFTTEELRNANVAMALYPLSAFR AMNRAAEKVYNVLRQEGTQKSVIDIMQTRNELYESINYYQ FEEKLDALYAKKS
82	prpB	Salmonella	ATGACGTTACACTCACCGGGTCAGGCGTTTCGCGCTGCG CTTGCTAAAGAAAAACCATTACAAATTGTCGGCGCTATC AACGCCAATCATGCTCTGTTAGCCCAGAGGGCTGGGTAT CAGGCTCTCTATCTCTCGGGCGGCGGTGTTGCCGCAGGC TCGCTGGGGCTACCGGATCTGGGCATCTCCACCCTTGAT GACGTATTGACCGATATCCGCCGTATCACCGACGTCTGC CCGCTGCCGCTGCTGGTGGATGCCGATATTGGCTTCGGA TCGTCGGCGTTTAACGTAGCGCGTACCGTGAAATCGATT TCCAAAGCCGGCGCCGCCGCGCTGCATATTGAAGATCA GATTGGCGCCAAGCGCTGCGGGCATCGGCCAAATAAAG

-292WO 2017/023818

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			CGATCGTCTCGAAAGAAGAGATGGTGGACCGGATCCAC GCGGCGGTGGATGCGCGGACCGATCCTGACTTTGTCATT ATGGCGCGTACCGATGCGCTGGCGGTTGAAGGCCTTGAT GCCGCTATCGATCGCGCGCGGGCCTACGTAGAGGCCGG TGCCGACATGCTGTTCCCGGAGGCGATTACTGAACTTGC GATGTACCGCCAGTTTGCCGACGCAGTGCAGGTGCCAAT CCTTGCCAATATTACCGAATTCGGCGCGACGCCGTTGTT TACTACCGAAGAGCTACGCAACGCCAACGTGGCGATGG CGCTCTATCCGCTGTCGGCGTTCCGGGCGATGAATCGCG CGGCGGAGAAGGTTTACAACGTGCTGCGACAGGAAGGA ACGCAAAAGAGCGTTATCGACATCATGCAGACCCGTAA TGAGCTGTATGAAAGCATCAATTATTACCAGTTCGAGGA AAAACTTGACGCGCTGTACGCCAAAAAATCGTAG
83	prpBCD	E. coli	atgtctctacactctccaggtaaagcgtttcgcgctgcacttagcaaagaaaccccgttgcaaa ttgttggcaccatcaacgctaaccatgcgctgctggcgcagcgtgccggatatcaggcgattt atctctccggcggtggcgtggcggcaggatcgctggggctgcccgatctcggtatttctactc ttgatgacgtgctgacagatattcgccgtatcaccgacgtttgttcgctgccgctgctggtggat gcggatatcggttttggttcttcagcctttaacgtggcgcgtacggtgaaatcaatgattaaagc cggtgcggcaggattgcatattgaagatcaggttggtgcgaaacgctgcggtcatcgtccga ataaagcgatcgtctcgaaagaagagatggtggatcggatccgcgcggcggtggatgcga aaaccgatcctgattttgtgatcatggcgcgcaccgatgcgctggcggtagaggggctggat gcggcgatcgagcgtgcgcaggcctatgttgaagcgggtgccgaaatgctgttcccggagg cgattaccgaactcgccatgtatcgccagtttgccgatgcggtgcaggtgccgatcctctcca acattaccgaatttggcgcaacaccgctgtttaccaccgacgaattacgcagcgcccatgtcg caatggcgctctacccgctttcagcgtttcgcgccatgaaccgcgccgctgaacatgtctataa catcctgcgtcaggaaggcacacagaaaagcgtcatcgacaccatgcagacccgcaacga gctgtacgaaagcatcaactactaccagtacgaagagaagctcgacgacctgtttgcccgtg gtcaggtgaaataaaaacgcccgttggttgtattcgacaaccgatgcctgatgcgccgctgac gcgacttatcaggcctacgaggtgaactgaactgtaggtcggataagacgcatagcgtcgca tccgacaacaatctcgaccctacaaatgataacaatgacgaggacaatatgagcgacacaac gatcctgcaaaacagtacccatgtcattaaaccgaaaaaatcggtggcactttccggcgttcc ggcgggcaatacggcgctctgcaccgtgggtaaaagcggcaacgacctgcattaccgtgg ctacgatattcttgatctggcggaacattgtgaatttgaagaagtggcgcacctgctgatccac ggcaaactgccaacccgtgacgaactcgccgcctacaaaacgaaactgaaagccctgcgtg gtttaccggctaacgtgcgtaccgtgctggaagccttaccggcggcgtcacacccgatggat gttatgcgcaccggcgtttccgcgctcggctgcacgctgccagaaaaagaggggcacaccg tttctggtgcgcgggatattgccgacaaactgctggcgtcacttagttcgattcttctctactggt atcactacagccacaacggcgaacgcatccagccggaaactgatgacgactctatcggcgg tcacttcctgcatctgctgcacggcgaaaagccgtcgcaaagctgggaaaaggcgatgcata tctcgctggtgctgtacgccgaacacgagtttaacgcttccacctttaccagccgggtgattgc gggcactggctctgatatgtattccgccattattggcgcgattggcgcactgcgcgggccgaa acacggcggggcgaatgaagtgtcgctggagatccagcaacgctacgaaacgccgggcg aagccgaagccgatatccgcaagcgggtggaaaacaaagaagtggtcattggttttgggcat ccggtttataccatcgccgacccgcgtcatcaggtgatcaaacgtgtggcgaagcagctctc gcaggaaggcggctcgctgaagatgtacaacatcgccgatcgcctggaaacggtgatgtgg gagagcaaaaagatgttccccaatctcgactggttctccgctgtttcctacaacatgatgggtgt tcccaccgagatgttcacaccactgtttgttatcgcccgcgtcactggctgggcggcgcacatt atcgaacaacgtcaggacaacaaaattatccgtccttccgccaattatgttggaccggaagac cgccagtttgtcgcgctggataagcgccagtaaacctctacgaataacaataaggaaacgta cccaatgtcagctcaaatcaacaacatccgcccggaatttgatcgtgaaatcgttgatatcgtc gattacgtgatgaactacgaaatcagctccagagtagcctacgacaccgctcattactgcctg cttgacacgctcggctgcggtctggaagctctcgaatatccggcctgtaaaaaactgctggg gccaattgtccccggcaccgtcgtacccaacggcgtgcgcgttcccggaactcagtttcagct cgaccccgtccaggcggcatttaacattggcgcgatgatccgttggctcgatttcaacgatac ctggctggcggcggagtgggggcatccttccgacaacctcggcggcattctggcaacggc ggactggctttcgcgcaacgcgatcgccagcggcaaagcgccgttgaccatgaaacaggtg ctgaccggaatgatcaaagcccatgaaattcagggctgcatcgcgctggaaaactcctttaac cgcgttggtctcgaccacgttctgttagtgaaagtggcttccaccgccgtggtcgccgaaatg

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			ctcggcctgacccgcgaggaaattctcaacgccgtttcgctggcatgggtagacggacagtc gctgcgcacttatcgtcatgcaccgaacaccggtacgcgtaaatcctgggcggcgggcgat gctacatcccgcgcggtacgtctggcgctgatggcgaaaacgggcgaaatgggttacccgt cagccctgaccgcgccggtgtggggtttctacgacgtctcctttaaaggtgagtcattccgctt ccagcgtccgtacggttcctacgtcatggaaaatgtgctgttcaaaatctccttcccggcggag ttccactcccagacggcagttgaagcggcgatgacgctctatgaacagatgcaggcagcag gcaaaacggcggcagatatcgaaaaagtgaccattcgcacccacgaagcctgtattcgcatc atcgacaaaaaagggccgctcaataacccggcagaccgcgaccactgcattcagtacatgg tggcgatcccgctgctgttcggacgcttaacggcggcagattacgaggacaacgttgcgcaa gataaacgcatcgacgccctgcgcgagaagatcaattgctttgaagatccggcgtttaccgct gactaccacgacccggaaaaacgcgccatcgccaatgccataacccttgagttcaccgacg gcacacgatttgaagaagtggtggtggagtacccaattggtcatgctcgccgccgtcaggat ggcattccgaagctggtcgataaattcaaaatcaatctcgcgcgccagttcccgactcgccag cagcagcgcattctggaggtttctctcgacagaactcgcctggaacagatgccggtcaatga gtatctcgacctgtacgtcatttaa
84	prpBCD	Salmonella	ATGACGTTACACTCACCGGGTCAGGCGTTTCGCGCTGCG CTTGCTAAAGAAAAACCATTACAAATTGTCGGCGCTATC AACGCCAATCATGCTCTGTTAGCCCAGAGGGCTGGGTAT CAGGCTCTCTATCTCTCGGGCGGCGGTGTTGCCGCAGGC TCGCTGGGGCTACCGGATCTGGGCATCTCCACCCTTGAT GACGTATTGACCGATATCCGCCGTATCACCGACGTCTGC CCGCTGCCGCTGCTGGTGGATGCCGATATTGGCTTCGGA TCGTCGGCGTTTAACGTAGCGCGTACCGTGAAATCGATT TCCAAAGCCGGCGCCGCCGCGCTGCATATTGAAGATCA GATTGGCGCCAAGCGCTGCGGGCATCGGCCAAATAAAG CGATCGTCTCGAAAGAAGAGATGGTGGACCGGATCCAC GCGGCGGTGGATGCGCGGACCGATCCTGACTTTGTCATT ATGGCGCGTACCGATGCGCTGGCGGTTGAAGGCCTTGAT GCCGCTATCGATCGCGCGCGGGCCTACGTAGAGGCCGG TGCCGACATGCTGTTCCCGGAGGCGATTACTGAACTTGC GATGTACCGCCAGTTTGCCGACGCAGTGCAGGTGCCAAT CCTTGCCAATATTACCGAATTCGGCGCGACGCCGTTGTT TACTACCGAAGAGCTACGCAACGCCAACGTGGCGATGG CGCTCTATCCGCTGTCGGCGTTCCGGGCGATGAATCGCG CGGCGGAGAAGGTTTACAACGTGCTGCGACAGGAAGGA ACGCAAAAGAGCGTTATCGACATCATGCAGACCCGTAA TGAGCTGTATGAAAGCATCAATTATTACCAGTTCGAGGA AAAACTTGACGCGCTGTACGCCAAAAAATCGTAGGCCA CGGGTCTGATAAAGCGTAGCCGCTATCAAGTCTGTGGCG GACAACCTCAATACCCTACACATTACAAAAATGACGAG GACACTATGAGCGACACGACGATCCTGCAAAACAACAC AAATGTCATTAAGCCAAAAAAATCCGTCGCATTATCCGG CGTACCCGCCGGAAATACCGCCTTATGCACCGTAGGTAA AAGCGGTAACGATCTGCACTATCGCGGGTACGATATTCT CGATCTCGCGGAGCACTGTGAATTTGAAGAAGTTGCGC ATCTGCTCATTCACGGCAAGCTGCCCACCCGTGATGAGC TGAATGCCTATAAAAGCAAATTAAAAGCGCTGCGTGGC TTACCCGCTAACGTCCGTACCGTGCTGGAAGCGCTGCCA GCGGCATCGCACCCGATGGACGTAATGCGCACCGGCGT TTCTGCGCTGGGCTGCACCCTGCCGGAAAAAGAGGGGC ATACCGTTTCTGGCGCGCGTGATATCGCCGACAAGCTGC TGGCCTCCCTCAGCTCCATTCTCCTTTACTGGTATCACTA CAGCCACAACGGCGAACGCATTCAGCCAGAAACTGACG ATGACTCTATCGGCGGGCATTTCCTGCATTTATTACACG GCGAAAAGCCATCGCAAAGCTGGGAAAAGGCGATGCAC ATTTCACTGGTACTGTACGCCGAACATGAGTTCAACGCC TCAACCTTTACCAGCCGGGTGGTAGCCGGTACGGGATCG GATATGTACTCCGCCATCATTGGCGCGATAGGCGCGCTT

-294WO 2017/023818

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			CGCGGGCCGAAGCACGGCGGGGCGAATGAAGTCTCGCT GGAGATTCAGCAGCGCTACGAAACGCCGGATGAAGCAG AAGCCGATATCCGTAAACGTATCGCCAATAAAGAAGTG GTGATTGGTTTTGGTCATCCGGTATACACCATCGCCGAT CCGCGCCATCAGGTGATTAAGCGGGTAGCGAAGCAGCT TTCACAGGAGGGCGGTTCGCTGAAGATGTACAACATTG CCGATCGGCTGGAGACGGTAATGTGGGACAGCAAAAAG ATGTTCCCTAATCTCGACTGGTTCTCGGCGGTCTCCTAC AACATGATGGGCGTTCCCACCGAAATGTTTACCCCGCTG TTTGTGATTGCCCGCGTTACAGGTTGGGCGGCGCACATC ATCGAGCAACGACAGGACAACAAAATTATCCGTCCTTC CGCCAATTATATTGGCCCGGAAGATCGCGCCTTTACGCC GCTGGAACAGCGTCAGTAAACCCTTACCTCTAACGATAA AAAGGAGTTGCACCCTATGTCCGCACCTGTTTCGAACGT CCGCCCTGAATTTGACCGTGAAATTGTTGATATTGTTGA TTATGTGATGAAGTACAACATCACCTCAAAAGTGGCTTA TGACACCGCGCACTACTGTCTGCTTGATACCCTGGGCTG TGGGCTGGAAGCGCTGGAATATCCGGCCTGTAAAAAAT TGATGGGGCCTATCGTGCCAGGTACCGTGGTGCCGAAC GGTGTACGTGTACCGGGCACTCAGTTCCAGCTCGATCCG GTGCAGGCGGCATTTAATATTGGCGCGATGATCCGCTGG CTCGACTTTAACGATACCTGGCTTGCCGCTGAGTGGGGA CACCCTTCCGATAACCTCGGCGGTATTCTGGCGACCGCC GACTGGTTGTCGCGCAACGCCGTCGCCGCCGGTAAAGC GCCGCTGACCATGCAGCAGGTGCTGACCGGGATGATCA AAGCCCACGAAATCCAGGGCTGTATCGCGCTGGAAAAC TCGTTTAACCGCGTGGGTCTCGATCACGTTTTGCTGGTG AAAGTGGCTTCCACGGCTGTAGTGGCTGAAATGCTCGGC CTGACCCGCGATGAAATTCTCAACGCCGTATCGCTGGCG TGGGTGGATGGGCAGTCGCTGCGTACCTATCGCCATGCG CCAAACACCGGTACGCGCAAATCCTGGGCGGCAGGCGA TGCCACTTCACGCGCGGTGCGTCTGGCGCTGATGGCGAA AACTGGCGAGATGGGCTATCCCTCGGCGTTGACCGCCA AAACCTGGGGCTTTTATGACGTCTCGTTCAAAGGCGAAA AATTCCGTTTCCAGCGCCCGTACGGCTCCTACGTGATGG AAAACGTGCTGTTCAAAATCTCCTTCCCGGCGGAGTTCC ATTCGCAGACCGCCGTTGAAGCAGCGATGACGCTGTAT GAGCAGATGCAGGCGGCTGGAAAAACGGCGGCGGATAT CGAAAAAGTAACGATTCGCACCCATGAAGCCTGTATAC GCATCATTGATAAAAAAGGCCCGCTGAATAATCCGGCT GACCGCGATCACTGTATTCAGTATATGGTGGCGATCCCA CTGCTGTTCGGACGCTTAACGGCGGCGGATTATGAGGAT GGCGTGGCGCAGGATAAACGTATTGACGCGCTGCGTGA AAAAACGCATTGCTTTGAAGACCCGGCGTTTACCACTGA TTATCATGACCCGGAAAAACGTTCGATTGCCAACGCCAT TAGTCTTGAATTTACTGACGGTACCCGTTTTGACGAGGT GGTTGTCGAGTACCCGATCGGCCACGCGCGTCGTCGCGG CGACGGCATTCCAAAACTTATCGAAAAATTTAAAATCA ATCTGGCGCGCCAGTTCCCACCCCGCCAGCAACAACGC ATCCTGGATGTCTCCCTGGACAGAACGCGCCTGGAGCA GATGCCGGTTAATGAGTATCTCGACTTGTACGTCATCTA G
85	PrpR	E. coli	MAHPPRLNDDKPVIWTVSVTRLFELFRDISLEFDHLANITPI QLGFEKAVAYIRKKLASERCDAIIAAGSNGAYLKSRLSVPV ILIKPSGYDVLQALAKAGKLTSSIGVVTYQETIPALVAFQKT FNLRLDQRSYITEEDARGQINELKANGTEAVVGAGLITDLA EEAGMTGIFIYSAATVRQAFSDALDMTRMSLRHNTHDATR NALRTRYVLGDMLGQSPQMEQVRQTILLYARSSAAVLIEG

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			ETGTGKELAAQAIHREYFARHDVRQGKKSHPFVAVNCGAI AESLLEAELFGYEEGAFTGSRRGGRAGLFEIAHGGTLFLDE IGEMPLPLQTRLLRVLEEKEVTRVGGHQPVPVDVRVISATH CNLEEDMQQGQFRRDLFYRLSILRLQLPPLRERVADILPLA ESFLKMSLAALSVPFSAALRQGLETCQIVLLLYDWPGNIRE LRNMMERLALFLSVEPTPDLTPQFLQLLLPELARESAKTPIP GLLTAQQALEKFNGDKTAAANYLGISRTTFWRRLKS
86	prpR	E. coli	tcagcttttcagccgccgccagaacgtcgtccggctgatacctaaataattcgccgctgctgtc ttatcgccattaaatttctccagtgcctgttgtgctgtcagcaagcctggaatgggagtcttcgc cgactcgcgcgccagttccggcagtagcagctgcaaaaattgcggcgttaaatccggcgtc ggttccacacttaaaaataacgccagtcgttccatcatattgcgcagttcacgaatattgcccgg ccagtcgtagagcaataatacaatctgacaggtctctaatccctgacgtaatgcggcagaaaa agggacagagagtgccgccagagacattttcaaaaagctttccgccagcggcagaatatcc gccacccgctcgcgtagcggcggcagttgcaggcgtaaaatactcagccgataaaacaggt cacggcgaaactgcccttgctgcatatcttcttccagattgcagtgagtggcgctaatgacccg cacatctaccggaacaggctgatgcccgccgacgcgggtgacctctttttcttccagcacccg taacaggcgagtctgcaacggcagcggcatttcgccaatctcatccagaaacagcgtaccgc cgtgggcaatttcgaacagcccggcgcgacctccgcgtcgcgagccggtgaacgccccttc ctcatagccaaacagctctgcttccagcagcgattcggcaatcgccccgcagttgacggcaa caaacggatgtgactttttgccctgtcgcacatcgtggcgggcaaaatattctcgatgaatcgc ctgggccgccagctctttgcccgtccccgtttccccctcaatcaacaccgccgcactggagc gggcatacagcaaaatagtctgccgcacctgttccatctgtggtgattgaccgagcatatcgc ccagcacgtaacgagtacgcagggcgttgcgggtggcatcgtgagtgttatggcgtaacga catgcgcgtcatatccagcgcatcgctaaatgcctggcgcacggtggcggcagaatagataa aaattccggtcattccggcttcttctgccagatcggtaatcagccccgcgccgaccaccgcttc ggtgccgttggcttttagctcgttaatctgcccgcgtgcgtcttcttcggtaatgtagctacgttg gtcgaggcgcaaattaaaggttttttgaaacgctaccagtgccggaatggtttcctgataggtg acaacgccgatagaagaggtgagttttccggcttttgccagcgcctgtaacacatcgtagcca ctcggttttatcagaatcaccggtaccgacaggcggcttttcaggtacgcaccgttagagcca gcggcaatgatggcgtcgcagcgttcgctggccagttttttgcggatgtaggccaccgcttttt caaagccaagctgaataggggtgatgttcgccagatgatcaaactcgaggctgatatcgcga aacagctcgaacagccgcgttacagataccgtccagataaccggtttgtcgtcattcagccgt ggtggatgtgccat
87	MctC	Corynebact erium	MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVG KSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAI SLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADV LSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVL LDIHEFKWQAVVVGIVGIVMIAYVLLGGMKGTTYVQMIK AVLLVGGVAIMTVLTFVKVSGGLTTLLNDAVEKHAASDY AATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAG LPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGY GAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISA VAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQ VRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASA NLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSG NDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPD NMDDLAAEMEVRSLTGVGVEKAVDH
88	mctC	Corynebact erium	ATGAATTCCACTATTCTCCTTGCACAAGACGCTGTTTCT GAGGGCGTCGGTAATCCGATTCTTAACATCAGTGTCTTC GTCGTCTTCATTATTGTGACGATGACCGTGGTGCTTCGC GTGGGCAAGAGCACCAGCGAATCCACCGACTTCTACAC CGGTGGTGCTTCCTTCTCCGGAACCCAGAACGGTCTGGC TATCGCAGGTGACTACCTGTCTGCAGCGTCCTTCCTCGG AATCGTTGGTGCAATTTCACTCAACGGTTACGACGGATT CCTTTACTCCATCGGCTTCTTCGTCGCATGGCTTGTTGCA CTGCTGCTCGTGGCAGAGCCACTTCGTAACGTGGGCCGC TTCACCATGGCTGACGTGCTGTCCTTCCGACTGCGTCAG AAACCAGTCCGCGTCGCTGCGGCCTGCGGTACCCTCGCG

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			GTTACCCTCTTTTACTTGATCGCTCAGATGGCTGGTGCA GGTTCGCTTGTGTCCGTTCTGCTGGACATCCACGAGTTC AAGTGGCAGGCAGTTGTTGTCGGTATCGTTGGCATTGTC ATGATCGCCTACGTTCTTCTTGGCGGTATGAAGGGCACC ACATACGTTCAGATGATTAAGGCAGTTCTGCTGGTCGGT GGCGTTGCCATTATGACCGTTCTGACCTTCGTCAAGGTG TCTGGTGGCCTGACCACCCTTTTAAATGACGCTGTTGAG AAGCACGCCGCTTCAGATTACGCTGCCACCAAGGGGTA CGATCCAACCCAGATCCTGGAGCCTGGTCTGCAGTACGG TGCAACTCTGACCACTCAGCTGGACTTCATTTCCTTGGC TCTCGCTCTGTGTCTTGGAACCGCTGGTCTGCCACACGT TCTGATGCGCTTCTACACCGTTCCTACCGCCAAGGAAGC ACGTAAGTCTGTGACCTGGGCTATCGTCCTCATTGGTGC GTTCTACCTGATGACCCTGGTCCTTGGTTACGGCGCTGC GGCACTGGTCGGTCCAGACCGCGTCATTGCCGCACCAG GTGCTGCTAATGCTGCTGCTCCTCTGCTGGCCTTCGAGC TTGGTGGTTCCATCTTCATGGCGCTGATTTCCGCAGTTGC GTTCGCTACCGTTCTCGCCGTGGTCGCAGGTCTTGCAAT TACCGCATCCGCTGCTGTTGGTCACGACATCTACAACGC TGTTATCCGCAACGGTCAGTCCACCGAAGCGGAGCAGG TCCGAGTATCCCGCATCACCGTTGTCGTCATTGGCCTGA TTTCCATTGTCCTGGGAATTCTTGCAATGACCCAGAACG TTGCGTTCCTCGTGGCCCTGGCCTTCGCAGTTGCAGCAT CCGCTAACCTGCCAACCATCCTGTACTCCCTGTACTGGA AGAAGTTCAACACCACCGGCGCTGTGGCCGCTATCTACA CCGGTCTCATCTCCGCGCTGCTGCTGATCTTCCTGTCCCC AGCAGTCTCCGGTAATGACAGCGCAATGGTTCCAGGTG CAGACTGGGCAATCTTCCCACTGAAGAACCCAGGCCTC GTCTCCATCCCACTGGCATTCATCGCTGGTTGGATCGGC ACTTTGGTTGGCAAGCCAGACAACATGGATGATCTTGCT GCCGAAATGGAAGTTCGTTCCCTCACCGGTGTCGGTGTT GAAAAGGCTGTTGATCACTAA
89	PutP_6	Virgibacill us sp.	MDLTTLITFIVYLLGMLAIGLIMYYRTNNLSDYVLGGRDLG PGVAALSAGASDMSGWLLLGLPGAIYASGMSEAWMGIGL AVGAYLNWQFVAKRLRVYTEVSNNSITIPDYFENRFKDNS HILRVISAIVILLFFTFYTSSGMVAGAKLFEASFGLQYETAL WIGAVVVVSYTLLGGFLAVAWTDFIQGILMFLALIVVPIVA LDQMGGWNQAVQAVGEINPSHLNMVEGVGIMAIISSLAW GLGYFGQPHIIVRFMALRSAKDVPKAKFIGTAWMILGLYG AIFTGFVGLAFISTQEVPILSEFGIQVVNENGLQMLADPEKIF IAFSQILFHPVVAGILLAAILSAIMSTVDSQLLVSSSAVAEDF YKAIFRKKATGKELVWVGRIATVIIAIVALIIAMNPDSSVLD LVSYAWAGFGAAFGPIIILSLFWKRITRNGALAGIIVGAITVI VWGDFLSGGIFDLYEIVPGFILNMIVTVIVSLIDKPNPDLEA DFDETVEKMKE
90	putP_6	Virgibacill us sp.	atggatcttacgacattaataacttttatagtatatctactagggatgttggcgattggcctcatca tgtattatcgaaccaataatttatcagattatgttcttggtggacgtgatcttggtccaggcgtagc tgcattgagtgctggtgcatcggatatgagtggttggctgttattaggtttgcctggagcgattta tgcatctggtatgtctgaagcttggatggggatcgggttagctgtaggtgcttatttaaattggc aatttgtagctaagcgattacgcgtttataccgaggtatcaaataattccattacgatcccagatt attttgaaaatcggtttaaagataactcacatattcttcgtgttatatctgctatcgtaattttgttatt cttcactttttatacatcttcaggaatggttgcaggagcaaaattatttgaggcttcattcggtctc caatacgaaactgctctgtggattggtgcggttgtagttgtatcttatacgttacttggaggatttc tagcggttgcatggacagactttattcaaggtattcttatgttccttgcactaattgttgttccaatc gtcgcattagatcaaatgggtggctggaatcaagcggtacaagctgttggtgaaattaatcctt cccacctcaatatggttgaaggtgttggaataatggcaattatttcatcacttgcttggggcttag gttattttggacagccacatattattgttcgttttatggcattacgttcggcgaaagatgttccgaa agcgaaatttattggaacagcttggatgattttaggactttatggagcaatctttactggttttgta

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			ggactagcatttatcagtacacaagaagtaccgattctgtctgaattcgggattcaagtagttaa tgagaatggtttacaaatgttagccgatcctgaaaagatatttattgctttctcccaaatactattc catccagtagttgccggtatcttactagcggcaatcttgtctgcaattatgagtaccgttgattca cagttacttgtatcatcttcagcggttgcagaagatttctataaagctattttccgtaaaaaagcta ctggtaaagagcttgtttgggttggacgtattgctacagtgataattgcgattgttgctttaattatt gcaatgaacccagatagctctgtattggatctagttagttatgcatgggctggatttggtgcagc atttggaccaattatcatcttgtcattattctggaagagaatcacaagaaatggtgcactagcgg gtatcattgtaggtgccattacggtaattgtatggggagactttctatctggaggtatctttgacct ctacgaaattgttccaggctttatcttaaatatgattgtcaccgttattgtgagtcttatcgataaac cgaatccagatttagaagctgactttgatgaaaccgtagaaaaaatgaaagaataa
91	MhpT	E. coli	MSTRTPSSSSSRLMLTIGLCFLVALMEGLDLQAAGIAAGGI AQAFALDKMQMGWIFSAGILGLLPGALVGGMLADRYGRK RILIGSVALFGLFSLATAIAWDFPSLVFARLMTGVGLGAAL PNLIALTSEAAGPRFRGTAVSLMYCGVPIGAALAATLGFAG ANLAWQTVFWVGGVVPLILVPLLMRWLPESAVFAGEKQS APPLRALFAPETATATLLLWLCYFFTLLVVYMLINWLPLLL VEQGFQPSQAAGVMFALQMGAASGTLMLGALMDKLRPV TMSLLIYSGMLASLLALGTVSSFNGMLLAGFVAGLFATGG QSVLYALAPLFYSSQIRATGVGTAVAVGRLGAMSGPLLAG KMLALGTGTVGVMAASAPGILVAGLAVFILMSRRSRIQPC ADA
92	mhpT	E. coli	atgTCGACTCGTACCCCTTCATCATCTTCATCCCGCCTGAT GCTGACCATCGGGCTTTGTTTTTTGGTCGCTCTGATGGA AGGGCTGGATCTTCAGGCGGCTGGCATTGCGGCGGGTG GCATCGCCCAGGCTTTCGCACTCGATAAAATGCAAATGG GCTGGATATTTAGCGCCGGAATACTCGGTTTGCTACCCG GCGCGTTGGTTGGCGGAATGCTGGCGGACCGTTATGGTC GCAAGCGCATTTTGATTGGCTCAGTTGCGCTGTTTGGTT TGTTCTCACTGGCAACGGCGATTGCCTGGGATTTCCCCT CACTGGTCTTTGCGCGGCTGATGACCGGTGTCGGGCTGG GGGCGGCGTTGCCGAATCTTATCGCCCTGACGTCTGAAG CCGCGGGTCCACGTTTTCGTGGGACGGCAGTGAGCCTGA TGTATTGCGGTGTTCCCATTGGCGCGGCGCTGGCGGCGA CACTGGGTTTCGCGGGGGCAAACTTAGCATGGCAAACG GTGTTTTGGGTAGGTGGTGTGGTGCCGTTGATTCTGGTG CCGCTATTAATGCGCTGGCTGCCGGAGTCGGCGGTTTTC GCTGGCGAAAAACAGTCTGCGCCACCACTGCGTGCCTTA TTTGCGCCAGAAACGGCAACCGCGACGCTGCTGCTGTG GTTGTGTTATTTCTTCACTCTGCTGGTGGTCTACATGTTG ATCAACTGGCTACCGCTACTTTTGGTGGAGCAAGGATTC CAGCCATCGCAGGCGGCAGGGGTGATGTTTGCCCTGCA AATGGGGGCGGCAAGCGGGACGTTAATGTTGGGCGCAT TGATGGATAAGCTGCGTCCAGTAACCATGTCGCTACTGA TTTATAGCGGCATGTTAGCTTCGCTGCTGGCGCTTGGAA CGGTGTCGTCATTTAACGGTATGTTGCTGGCGGGATTTG TCGCGGGGTTGTTTGCGACAGGTGGGCAAAGCGTTTTGT ATGCCCTGGCACCGTTGTTTTACAGTTCGCAGATCCGCG CAACAGGTGTGGGAACAGCCGTGGCGGTAGGGCGTCTG GGGGCTATGAGCGGTCCGTTACTGGCCGGGAAAATGCT GGCATTAGGCACTGGCACGGTCGGCGTAATGGCCGCTTC TGCACCGGGTATTCTTGTTGCTGGGTTGGCGGTGTTTATT TTGATGAGCCGGAGATCACGAATACAGCCGTGCGCCGA TGCCTGA
93	prpBCDE	E. coli	atgtctctacactctccaggtaaagcgtttcgcgctgcacttagcaaagaaaccccgttgcaaa ttgttggcaccatcaacgctaaccatgcgctgctggcgcagcgtgccggatatcaggcgattt atctctccggcggtggcgtggcggcaggatcgctggggctgcccgatctcggtatttctactc ttgatgacgtgctgacagatattcgccgtatcaccgacgtttgttcgctgccgctgctggtggat gcggatatcggttttggttcttcagcctttaacgtggcgcgtacggtgaaatcaatgattaaagc

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cggtgcggcaggattgcatattgaagatcaggttggtgcgaaacgctgcggtcatcgtccga ataaagcgatcgtctcgaaagaagagatggtggatcggatccgcgcggcggtggatgcga aaaccgatcctgattttgtgatcatggcgcgcaccgatgcgctggcggtagaggggctggat gcggcgatcgagcgtgcgcaggcctatgttgaagcgggtgccgaaatgctgttcccggagg cgattaccgaactcgccatgtatcgccagtttgccgatgcggtgcaggtgccgatcctctcca acattaccgaatttggcgcaacaccgctgtttaccaccgacgaattacgcagcgcccatgtcg caatggcgctctacccgctttcagcgtttcgcgccatgaaccgcgccgctgaacatgtctataa catcctgcgtcaggaaggcacacagaaaagcgtcatcgacaccatgcagacccgcaacga gctgtacgaaagcatcaactactaccagtacgaagagaagctcgacgacctgtttgcccgtg gtcaggtgaaataaaaacgcccgttggttgtattcgacaaccgatgcctgatgcgccgctgac gcgacttatcaggcctacgaggtgaactgaactgtaggtcggataagacgcatagcgtcgca tccgacaacaatctcgaccctacaaatgataacaatgacgaggacaatatgagcgacacaac gatcctgcaaaacagtacccatgtcattaaaccgaaaaaatcggtggcactttccggcgttcc ggcgggcaatacggcgctctgcaccgtgggtaaaagcggcaacgacctgcattaccgtgg ctacgatattcttgatctggcggaacattgtgaatttgaagaagtggcgcacctgctgatccac ggcaaactgccaacccgtgacgaactcgccgcctacaaaacgaaactgaaagccctgcgtg gtttaccggctaacgtgcgtaccgtgctggaagccttaccggcggcgtcacacccgatggat gttatgcgcaccggcgtttccgcgctcggctgcacgctgccagaaaaagaggggcacaccg tttctggtgcgcgggatattgccgacaaactgctggcgtcacttagttcgattcttctctactggt atcactacagccacaacggcgaacgcatccagccggaaactgatgacgactctatcggcgg tcacttcctgcatctgctgcacggcgaaaagccgtcgcaaagctgggaaaaggcgatgcata tctcgctggtgctgtacgccgaacacgagtttaacgcttccacctttaccagccgggtgattgc gggcactggctctgatatgtattccgccattattggcgcgattggcgcactgcgcgggccgaa acacggcggggcgaatgaagtgtcgctggagatccagcaacgctacgaaacgccgggcg aagccgaagccgatatccgcaagcgggtggaaaacaaagaagtggtcattggttttgggcat ccggtttataccatcgccgacccgcgtcatcaggtgatcaaacgtgtggcgaagcagctctc gcaggaaggcggctcgctgaagatgtacaacatcgccgatcgcctggaaacggtgatgtgg gagagcaaaaagatgttccccaatctcgactggttctccgctgtttcctacaacatgatgggtgt tcccaccgagatgttcacaccactgtttgttatcgcccgcgtcactggctgggcggcgcacatt atcgaacaacgtcaggacaacaaaattatccgtccttccgccaattatgttggaccggaagac cgccagtttgtcgcgctggataagcgccagtaaacctctacgaataacaataaggaaacgta cccaatgtcagctcaaatcaacaacatccgcccggaatttgatcgtgaaatcgttgatatcgtc gattacgtgatgaactacgaaatcagctccagagtagcctacgacaccgctcattactgcctg cttgacacgctcggctgcggtctggaagctctcgaatatccggcctgtaaaaaactgctggg gccaattgtccccggcaccgtcgtacccaacggcgtgcgcgttcccggaactcagtttcagct cgaccccgtccaggcggcatttaacattggcgcgatgatccgttggctcgatttcaacgatac ctggctggcggcggagtgggggcatccttccgacaacctcggcggcattctggcaacggc ggactggctttcgcgcaacgcgatcgccagcggcaaagcgccgttgaccatgaaacaggtg ctgaccggaatgatcaaagcccatgaaattcagggctgcatcgcgctggaaaactcctttaac cgcgttggtctcgaccacgttctgttagtgaaagtggcttccaccgccgtggtcgccgaaatg ctcggcctgacccgcgaggaaattctcaacgccgtttcgctggcatgggtagacggacagtc gctgcgcacttatcgtcatgcaccgaacaccggtacgcgtaaatcctgggcggcgggcgat gctacatcccgcgcggtacgtctggcgctgatggcgaaaacgggcgaaatgggttacccgt cagccctgaccgcgccggtgtggggtttctacgacgtctcctttaaaggtgagtcattccgctt ccagcgtccgtacggttcctacgtcatggaaaatgtgctgttcaaaatctccttcccggcggag ttccactcccagacggcagttgaagcggcgatgacgctctatgaacagatgcaggcagcag gcaaaacggcggcagatatcgaaaaagtgaccattcgcacccacgaagcctgtattcgcatc atcgacaaaaaagggccgctcaataacccggcagaccgcgaccactgcattcagtacatgg tggcgatcccgctgctgttcggacgcttaacggcggcagattacgaggacaacgttgcgcaa gataaacgcatcgacgccctgcgcgagaagatcaattgctttgaagatccggcgtttaccgct gactaccacgacccggaaaaacgcgccatcgccaatgccataacccttgagttcaccgacg gcacacgatttgaagaagtggtggtggagtacccaattggtcatgctcgccgccgtcaggat ggcattccgaagctggtcgataaattcaaaatcaatctcgcgcgccagttcccgactcgccag cagcagcgcattctggaggtttctctcgacagaactcgcctggaacagatgccggtcaatga gtatctcgacctgtacgtcatttaagtaaacggcggtaaggcgtaagttcaacaggagagcatt atgtcttttagcgaattttatcagcgttcgattaacgaaccggagaagttctgggccgagcagg cccggcgtattgactggcagacgccctttacgcaaacgctcgaccacagcaacccgccgttt gcccgttggttttgtgaaggccgaaccaacttgtgtcacaacgctatcgaccgctggctggag

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			aaacagccagaggcgctggcattgattgccgtctcttcggaaacagaggaagagcgtacctt taccttccgccagttacatgacgaagtgaatgcggtggcgtcaatgctgcgctcactgggcgt gcagcgtggcgatcgggtgctggtgtatatgccgatgattgccgaagcgcatattaccctgct ggcctgcgcgcgcattggtgctattcactcggtggtgtttgggggatttgcttcgcacagcgtg gcaacgcgaattgatgacgctaaaccggtgctgattgtctcggctgatgccggggcgcgcg gcggtaaaatcattccgtataaaaaattgctcgacgatgcgataagtcaggcacagcatcagc cgcgtcacgttttactggtggatcgcgggctggcgaaaatggcgcgcgttagcgggcggga tgtcgatttcgcgtcgttgcgccatcaacacatcggcgcgcgggtgccggtggcatggctgg aatccaacgaaacctcctgcattctctacacctccggcacgaccggcaaacctaaaggtgtg cagcgtgatgtcggcggatatgcggtggcgctggcgacctcgatggacaccatttttggcgg caaagcgggcggcgtgttcttttgtgcttcggatatcggctgggtggtagggcattcgtatatc gtttacgcgccgctgctggcggggatggcgactatcgtttacgaaggattgccgacctggcc ggactgcggcgtgtggtggaaaattgtcgagaaatatcaggttagccgcatgttctcagcgcc gaccgccattcgcgtgctgaaaaaattccctaccgctgaaattcgcaaacacgatctttcgtcg ctggaagtgctctatctggctggagaaccgctggacgagccgaccgccagttgggtgagca atacgctggatgtgccggtcatcgacaactactggcagaccgaatccggctggccgattatg gcgattgctcgcggtctggatgacagaccgacgcgtctgggaagccccggcgtgccgatgt atggctataacgtgcagttgctcaatgaagtcaccggcgaaccgtgtggcgtcaatgagaaa gggatgctggtagtggaggggccattgccgccaggctgtattcaaaccatctggggcgacg acgaccgctttgtgaagacgtactggtcgctgttttcccgtccggtgtacgccacttttgactgg ggcatccgcgatgctgacggttatcactttattctcgggcgcactgacgatgtgattaacgttg ccggacatcggctgggtacgcgtgagattgaagagagtatctccagtcatccgggcgttgcc gaagtggcggtggttggggtgaaagatgcgctgaaagggcaggtggcggtggcgtttgtca ttccgaaagagagcgacagtctggaagaccgtgaggtggcgcactcgcaagagaaggcga ttatggcgctggtggacagccagattggcaactttggccgcccggcgcacgtctggtttgtct cgcaattgccaaaaacgcgatccggaaaaatgctgcgccgcacgatccaggcgatttgcga aggacgcgatcctggggatctgacgaccattgatgatccggcgtcgttggatcagatccgcc aggcgatggaagagtag
94	prpBCDE	Salmonella	ATGACGTTACACTCACCGGGTCAGGCGTTTCGCGCTGCG CTTGCTAAAGAAAAACCATTACAAATTGTCGGCGCTATC AACGCCAATCATGCTCTGTTAGCCCAGAGGGCTGGGTAT CAGGCTCTCTATCTCTCGGGCGGCGGTGTTGCCGCAGGC TCGCTGGGGCTACCGGATCTGGGCATCTCCACCCTTGAT GACGTATTGACCGATATCCGCCGTATCACCGACGTCTGC CCGCTGCCGCTGCTGGTGGATGCCGATATTGGCTTCGGA TCGTCGGCGTTTAACGTAGCGCGTACCGTGAAATCGATT TCCAAAGCCGGCGCCGCCGCGCTGCATATTGAAGATCA GATTGGCGCCAAGCGCTGCGGGCATCGGCCAAATAAAG CGATCGTCTCGAAAGAAGAGATGGTGGACCGGATCCAC GCGGCGGTGGATGCGCGGACCGATCCTGACTTTGTCATT ATGGCGCGTACCGATGCGCTGGCGGTTGAAGGCCTTGAT GCCGCTATCGATCGCGCGCGGGCCTACGTAGAGGCCGG TGCCGACATGCTGTTCCCGGAGGCGATTACTGAACTTGC GATGTACCGCCAGTTTGCCGACGCAGTGCAGGTGCCAAT CCTTGCCAATATTACCGAATTCGGCGCGACGCCGTTGTT TACTACCGAAGAGCTACGCAACGCCAACGTGGCGATGG CGCTCTATCCGCTGTCGGCGTTCCGGGCGATGAATCGCG CGGCGGAGAAGGTTTACAACGTGCTGCGACAGGAAGGA ACGCAAAAGAGCGTTATCGACATCATGCAGACCCGTAA TGAGCTGTATGAAAGCATCAATTATTACCAGTTCGAGGA AAAACTTGACGCGCTGTACGCCAAAAAATCGTAGGCCA CGGGTCTGATAAAGCGTAGCCGCTATCAAGTCTGTGGCG GACAACCTCAATACCCTACACATTACAAAAATGACGAG GACACTATGAGCGACACGACGATCCTGCAAAACAACAC AAATGTCATTAAGCCAAAAAAATCCGTCGCATTATCCGG CGTACCCGCCGGAAATACCGCCTTATGCACCGTAGGTAA AAGCGGTAACGATCTGCACTATCGCGGGTACGATATTCT CGATCTCGCGGAGCACTGTGAATTTGAAGAAGTTGCGC

-300WO 2017/023818

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ATCTGCTCATTCACGGCAAGCTGCCCACCCGTGATGAGC TGAATGCCTATAAAAGCAAATTAAAAGCGCTGCGTGGC TTACCCGCTAACGTCCGTACCGTGCTGGAAGCGCTGCCA GCGGCATCGCACCCGATGGACGTAATGCGCACCGGCGT TTCTGCGCTGGGCTGCACCCTGCCGGAAAAAGAGGGGC ATACCGTTTCTGGCGCGCGTGATATCGCCGACAAGCTGC TGGCCTCCCTCAGCTCCATTCTCCTTTACTGGTATCACTA CAGCCACAACGGCGAACGCATTCAGCCAGAAACTGACG ATGACTCTATCGGCGGGCATTTCCTGCATTTATTACACG GCGAAAAGCCATCGCAAAGCTGGGAAAAGGCGATGCAC ATTTCACTGGTACTGTACGCCGAACATGAGTTCAACGCC TCAACCTTTACCAGCCGGGTGGTAGCCGGTACGGGATCG GATATGTACTCCGCCATCATTGGCGCGATAGGCGCGCTT CGCGGGCCGAAGCACGGCGGGGCGAATGAAGTCTCGCT GGAGATTCAGCAGCGCTACGAAACGCCGGATGAAGCAG AAGCCGATATCCGTAAACGTATCGCCAATAAAGAAGTG GTGATTGGTTTTGGTCATCCGGTATACACCATCGCCGAT CCGCGCCATCAGGTGATTAAGCGGGTAGCGAAGCAGCT TTCACAGGAGGGCGGTTCGCTGAAGATGTACAACATTG CCGATCGGCTGGAGACGGTAATGTGGGACAGCAAAAAG ATGTTCCCTAATCTCGACTGGTTCTCGGCGGTCTCCTAC AACATGATGGGCGTTCCCACCGAAATGTTTACCCCGCTG TTTGTGATTGCCCGCGTTACAGGTTGGGCGGCGCACATC ATCGAGCAACGACAGGACAACAAAATTATCCGTCCTTC CGCCAATTATATTGGCCCGGAAGATCGCGCCTTTACGCC GCTGGAACAGCGTCAGTAAACCCTTACCTCTAACGATAA AAAGGAGTTGCACCCTATGTCCGCACCTGTTTCGAACGT CCGCCCTGAATTTGACCGTGAAATTGTTGATATTGTTGA TTATGTGATGAAGTACAACATCACCTCAAAAGTGGCTTA TGACACCGCGCACTACTGTCTGCTTGATACCCTGGGCTG TGGGCTGGAAGCGCTGGAATATCCGGCCTGTAAAAAAT TGATGGGGCCTATCGTGCCAGGTACCGTGGTGCCGAAC GGTGTACGTGTACCGGGCACTCAGTTCCAGCTCGATCCG GTGCAGGCGGCATTTAATATTGGCGCGATGATCCGCTGG CTCGACTTTAACGATACCTGGCTTGCCGCTGAGTGGGGA CACCCTTCCGATAACCTCGGCGGTATTCTGGCGACCGCC GACTGGTTGTCGCGCAACGCCGTCGCCGCCGGTAAAGC GCCGCTGACCATGCAGCAGGTGCTGACCGGGATGATCA AAGCCCACGAAATCCAGGGCTGTATCGCGCTGGAAAAC TCGTTTAACCGCGTGGGTCTCGATCACGTTTTGCTGGTG AAAGTGGCTTCCACGGCTGTAGTGGCTGAAATGCTCGGC CTGACCCGCGATGAAATTCTCAACGCCGTATCGCTGGCG TGGGTGGATGGGCAGTCGCTGCGTACCTATCGCCATGCG CCAAACACCGGTACGCGCAAATCCTGGGCGGCAGGCGA TGCCACTTCACGCGCGGTGCGTCTGGCGCTGATGGCGAA AACTGGCGAGATGGGCTATCCCTCGGCGTTGACCGCCA AAACCTGGGGCTTTTATGACGTCTCGTTCAAAGGCGAAA AATTCCGTTTCCAGCGCCCGTACGGCTCCTACGTGATGG AAAACGTGCTGTTCAAAATCTCCTTCCCGGCGGAGTTCC ATTCGCAGACCGCCGTTGAAGCAGCGATGACGCTGTAT GAGCAGATGCAGGCGGCTGGAAAAACGGCGGCGGATAT CGAAAAAGTAACGATTCGCACCCATGAAGCCTGTATAC GCATCATTGATAAAAAAGGCCCGCTGAATAATCCGGCT GACCGCGATCACTGTATTCAGTATATGGTGGCGATCCCA CTGCTGTTCGGACGCTTAACGGCGGCGGATTATGAGGAT GGCGTGGCGCAGGATAAACGTATTGACGCGCTGCGTGA AAAAACGCATTGCTTTGAAGACCCGGCGTTTACCACTGA TTATCATGACCCGGAAAAACGTTCGATTGCCAACGCCAT

-301WO 2017/023818

PCT/US2016/044922

			TAGTCTTGAATTTACTGACGGTACCCGTTTTGACGAGGT GGTTGTCGAGTACCCGATCGGCCACGCGCGTCGTCGCGG CGACGGCATTCCAAAACTTATCGAAAAATTTAAAATCA ATCTGGCGCGCCAGTTCCCACCCCGCCAGCAACAACGC ATCCTGGATGTCTCCCTGGACAGAACGCGCCTGGAGCA GATGCCGGTTAATGAGTATCTCGACTTGTACGTCATCTA GAACCTGTCTCATTAGGCGTAAGTTCTACAGGAGAGCAT TATGTCTTTTAGCGAATTTTATCAGCGTTCGATTAACGA ACCGGAGCAGTTCTGGGCTGAACAGGCCCGGCGTATCG ACTGGCAGCAGCCGTTTACGCAGACGCTGGACTACAGC AACCCGCCGTTTGCCCGCTGGTTTTGCGGCGGCACCACT AATCTGTGCCATAACGCGATTGACCGCTGGCTGGATACC CAGCCGGATGCGCTGGCGCTGATTGCGGTTTCCTCTGAG ACCGAAGAAGAACGTACCTTCACCTTTCGTCAACTGTAT GACGAGGTGAATGTCGTGGCCTCTATGCTGCTGTCACTG GGCGTGCGGCGTGGCGATCGGGTACTGGTGTATATGCC GATGATTGCCGAGGCGCACATCACATTACTGGCCTGCGC GCGCATTGGCGCGATCCATTCAGTGGTGTTTGGTGGTTT TGCCTCGCACAGTGTAGCCGCGCGCATCGACGATGCCA GACCGGTGCTGATTGTCTCGGCGGACGCCGGAGCGCGA GGTGGGAAGGTCATTCCCTATAAAAAGCTTCTTGATGAG GCGGTCGATCAGGCACAGCATCAGCCGAAGCATGTACT GCTGGTGGATCGGGGGCTGGCGAAAATGGCGCGGGTTG CCGGGCGCGATGTGGATTTTGCGACCCTGCGCGAACACC ATGCCGGGGCGCGTGTGCCAGTGGCCTGGCTTGAATCTA ATGAAAGTTCCTGCATTCTTTATACCTCCGGCACTACCG GCAAACCGAAAGGCGTTCAGCGTGACGTTGGTGGCTAC GCCGTGGCGCTGGCGACATCGATGGACACCCTCTTTGGC GGCAAAGCGGGCGGCGTCTTTTTCTGCGCTTCGGATATC GGTTGGGTAGTGGGGCACTCTTATATTGTGTATGCGCCG CTGCTGGCGGGTATGGCGACCATCGTTTATGAAGGATTG CCGACGTATCCGGACTGCGGCGTATGGTGGAAAATTGTC GAGAAATATCGGGTGAGCCGGATGTTTTCAGCGCCAAC CGCCATTCGTGTGCTGAAGAAATTTCCCACCGCGCAGAT ACGCAATCATGATCTCTCCTCGCTGGAAGTTCTCTATCT GGCAGGCGAGCCGCTCGACGAGCCAACGGCAGCCTGGG TTAGCGGAACACTGGGTGTGCCGGTGATCGACAATTACT GGCAGACCGAATCCGGCTGGCCGATTATGGCGCTGGCG CGCACGCTTGATGACAGACCATCGCGTTTGGGCAGTCCC GGCGTGCCGATGTACGGCTATAATGTTCAACTGCTCAAC GAGGTGACCGGTGAACCCTGTGGTGCGAACGAAAAGGG AATGGTGGTTATTGAAGGGCCGCTGCCGCCGGGCTGCAT TCAGACCATCTGGGGCGATGACGCACGCTTTGTGAATAC CTACTGGTCACTGTTTACTCGTCAGGTGTATGCCACCTTT GACTGGGGGATCCGCGACGCCGACGGCTATTATTTTATC CTTGGGCGCACGGATGATGTGATCAACGTCGCCGGACA TCGTCTCGGCACCCGTGAGATAGAGGAGAGCATCTCCA GCTATCCCAACGTTGCGGAAGTGGCGGTGGTAGGGGTA AAAGACGCGCTGAAAGGGCAGGTAGCGGTAGCCTTCGT GATCCCGAAACAGAGTGACAGTCTGGAAGACCGCGAAG TGGCGCATTCGGAAGAGAAGGCGATTATGGCGCTGGTC GATAGTCAGATCGGCAACTTTGGCCGCCCGGCGCACGT GTGGTTTGTCTCGCAGCTACCAAAAACCCGATCCGGGAA GATGCTCAGACGAACGATCCAGGCGATCTGCGAGGGCC GGGATCCAGGCGATCTGACGACCATTGACGATCCGACG TCGTTGCAACAAATTCGCCAGGTCATTGAGGAGTAA
95	PccB	Bifidobact eriurn	MTDIMDSQAVKAAAAASAANAAQPSAHQPLRTAVVKAA ELARAAEERARDKQHAKGKKTARERLDLLFDTGTFEEIGR

-302WO 2017/023818

PCT/US2016/044922

		longum	FQGGNIAGGNAGAAVITGFGQVYGRKVAVYAQDFSVKGG TLGTAEGEKICRLMDMAIDLKVPIVAIVDSGGARIQEGVAA LTQYGRIFRKTCEASGFVPQLSLILGPCAGGAVYCPALTDLI IMTRENSNMFVTGPDVVKASTGETISMADLGGGEVHNRVS GVAHYLGEDESDAIDYARTVLAYLPSNSESKPPVYAYAVT RAERETAKRLATIVPTNERQPYDMLEVIRCIVDYGEFVQVQ ELFGASALVGFACIDGKPVGIVANQPNVLAGILDVDSSEKV ARFVRLCDAFNLPVVTLVDVPGYKPGSDQEHAGIIRRGAK VIYAYANAQVPMVTVVLRKAFGGAYIVMGSKAIGADLNF AWPSSQIAVLGAAGAVNIIHRHDLAKAKASGQDVDALRA KYIKEYETSTVNANLSLEIGQIDGMIDPEQTREVIVESLATL ATKRRVKRTTKHHGNQPL
96	pccB	Bifidobact eriurn longum	TCAGAGGGGCTGGTTGCCGTGGTGTTTGGTGGTGCGCTT GACGCGCCGCTTGGTGGCGAGCGTGGCCAGCGATTCGA CAATCACCTCACGGGTCTGTTCGGGGTCGATCATGCCGT CGATCTGCCCGATTTCCAGTGACAGGTTCGCGTTGACGG TGCTGGTCTCGTACTCCTTGATGTACTTGGCCCGCAGCG CATCGACGTCCTGTCCGGAGGCCTTGGCCTTGGCCAGGT CGTGGCGGTGGATGATGTTCACCGCGCCGGCCGCGCCG AGCACCGCGATCTGGGAGGAGGGCCACGCGAAGTTCAG GTCCGCGCCAATGGCCTTGGATCCCATCACGATGTACGC GCCGCCGAACGCCTTGCGCAACACCACGGTCACCATCG GTACCTGTGCGTTGGCGTAGGCGTAGATCACCTTGGCGC CGCGGCGGATGATGCCGGCGTGTTCCTGGTCGGAGCCG GGCTTGTAGCCGGGCACATCCACGAGGGTGACCACGGG CAGGTTGAACGCGTCGCACAGGCGTACGAATCGGGCGA CTTTCTCGGACGAGTCGACGTCCAGGATGCCGGCGAGC ACGTTCGGCTGGTTCGCCACGATGCCAACCGGCTTGCCG TCGATGCAGGCGAAGCCGACGAGCGCGGAGGCGCCGAA CAGTTCCTGCACCTGCACGAATTCGCCGTAATCGACGAT GCAACGAATCACTTCGAGCATGTCGTAAGGCTGACGTTC GTTGGTGGGCACGATGGTGGCAAGTCGCTTGGCGGTCTC GCGTTCGGCGCGGGTGACGGCGTATGCGTAGACCGGCG GCTTGCTTTCGCTGTTGGACGGCAGGTAGGCGAGCACGG TGCGCGCATAGTCGATGGCGTCGGATTCGTCCTCGCCGA GGTAGTGGGCCACGCCGGACACCCGGTTGTGCACTTCGC CGCCGCCGAGGTCGGCCATGGAGATGGTCTCGCCGGTC GAGGCCTTGACCACGTCCGGTCCGGTGACGAACATGTTC GAGTTCTCACGGGTCATGATGATGAGGTCCGTCAGGGCC GGGCAGTAGACGGCACCGCCGGCGCAGGGGCCGAGAAT CAGGCTCAGCTGGGGCACGAAGCCGCTGGCCTCGCAAG TCTTGCGGAAGATGCGACCGTACTGGGTCAGGGCGGCC ACGCCCTCCTGGATGCGGGCGCCGCCGGAGTCCACGAT GGCCACGATCGGCACTTTGAGGTCGATGGCCATGTCCAT CAGTCGGCAGATCTTCTCGCCTTCGGCGGTGCCGAGGGT GCCGCCCTTGACGGAGAAGTCCTGGGCGTAGACGGCCA CTTTGCGGCCGTAGACCTGGCCGAAGCCGGTGATGACG GCCGCACCGGCGTTGCCGCCGGCGATATTGCCGCCCTGG AAGCGGCCGATCTCCTCGAACGTGCCGGTGTCGAAGAG CAGGTCGAGGCGTTCGCGCGCGGTTTTCTTGCCTTTGGC GTGCTGCTTGTCGCGGGCGCGCTCTTCGGCGGCGCGGGC CAGTTCGGCGGCCTTGACCACAGCGGTGCGCAGCGGCT GGTGGGCCGAAGGCTGGGCGGCGTTGGCGGCCGAGGCC GCAGCCGCGGCCTTCACGGCCTGCGAATCCATGATGTCA

-303WO 2017/023818

PCT/US2016/044922

			GTCAT
97	GltA	E. coli	MADTKAKLTLNGDTAVELDVLKGTLGQDVIDIRTLGSKG VFTFDPGFTSTASCESKITFIDGDEGILLHRGFPIDQLATDSN YLEVCYILLNGEKPTQEQYDEFKTTVTRHTMIHEQITRLFH AFRRDSHPMAVMCGITGALAAFYHDSLDVNNPRHREIAAF RLLSKMPTMAAMCYKYSIGQPFVYPRNDLSYAGNFLNMM FSTPCEPYEVNPILERAMDRILILHADHEQNASTSTVRTAGS SGANPFACIAAGIASLWGPAHGGANEAALKMLEEISSVKHI PEFVRRAKDKNDSFRLMGFGHRVYKNYDPRATVMRETCH EVLKELGTKDDLLEVAMELENIALNDPYFIEKKLYPNVDF YSGIILKAMGIPSSMFTVIFAMARTVGWIAHWSEMHSDGM KIARPRQLYTGYEKRDFKSDIKR
98	g//A	E. coli	ATGGCTGATACAAAAGCAAAACTCACCCTCAACGGGGA TACAGCTGTTGAACTGGATGTGCTGAAAGGCACGCTGG GTCAAGATGTTATTGATATCCGTACTCTCGGTTCAAAAG GTGTGTTCACCTTTGACCCAGGCTTCACTTCAACCGCAT CCTGCGAATCTAAAATTACTTTTATTGATGGTGATGAAG GTATTTTGCTGCACCGCGGTTTCCCGATCGATCAGCTGG CGACCGATTCTAACTACCTGGAAGTTTGTTACATCCTGC TGAATGGTGAAAAACCGACTCAGGAACAGTATGACGAA TTTAAAACTACGGTGACCCGTCATACCATGATCCACGAG CAGATTACCCGTCTGTTCCATGCTTTCCGTCGCGACTCG CATCCAATGGCAGTCATGTGTGGTATTACCGGCGCGCTG GCGGCGTTCTATCACGACTCGCTGGATGTTAACAATCCT CGTCACCGTGAAATTGCCGCGTTCCGCCTGCTGTCGAAA ATGCCGACCATGGCCGCGATGTGTTACAAGTATTCCATT GGTCAGCCATTTGTTTACCCGCGCAACGATCTCTCCTAC GCCGGTAACTTCCTGAATATGATGTTCTCCACGCCGTGC GAACCGTATGAAGTTAATCCGATTCTGGAACGTGCTATG GACCGTATTCTGATCCTGCACGCTGACCATGAACAGAAC GCCTCTACCTCCACCGTGCGTACCGCTGGCTCTTCGGGT GCGAACCCGTTTGCCTGTATCGCAGCAGGTATTGCTTCA CTGTGGGGACCTGCGCACGGCGGTGCTAACGAAGCGGC GCTGAAAATGCTGGAAGAAATCAGCTCCGTTAAACACA TTCCGGAATTTGTTCGTCGTGCGAAAGACAAAAATGATT CTTTCCGCCTGATGGGCTTCGGTCACCGCGTGTACAAAA ATTACGACCCGCGCGCCACCGTAATGCGTGAAACCTGCC ATGAAGTGCTGAAAGAGCTGGGCACGAAGGATGACCTG CTGGAAGTGGCTATGGAGCTGGAAAACATCGCGCTGAA CGACCCGTACTTTATCGAGAAGAAACTGTACCCGAACGT CGATTTCTACTCTGGTATCATCCTGAAAGCGATGGGTAT TCCGTCTTCCATGTTCACCGTCATTTTCGCAATGGCACGT ACCGTTGGCTGGATCGCCCACTGGAGCGAAATGCACAG TGACGGTATGAAGATTGCCCGTCCGCGTCAGCTGTATAC AGGATATGAAAAACGCGACTTTAAAAGCGATATCAAGC GTTAA
99	PhaA	Acinetoba cter sp.	MKDVVIVAAKRTAIGSFLGSLASLSAPQLGQTAIRAVLDSA NVKPEQVDQVIMGNVLTTGVGQNPARQAAIAAGIPVQVP ASTLNVVCGSGLRAVHLAAQAIQCDEADIVVAGGQESMS QSAHYMQLRNGQKMGNAQLVDSMVADGLTDAYNQYQM GITAENIVEKLGLNREEQDQLALTSQQRAAAAQAAGKFKD EIAVVSIPQRKGEPVVFAEDEYIKANTSLESLTKLRPAFKKD GSVTAGNASGINDGAAAVLMMSADKAAELGLKPLARIKG YAMSGIEPEIMGLGPVDAVKKTLNKAGWSLDQVDLIEANE AFAAQALGVAKELGLDLDKVNVNGGAIALGHPIGASGCRI

-304WO 2017/023818

PCT/US2016/044922

			LVTLLHEMQRRDAKKGIATLCVGGGMGVALAVERD
100	PhaB	Acinetoba cter sp.	MSEQKVALVTGALGGIGSEICRQLVTAGYKIIATVVPREED REKQWLQSEGFQDSDVRFVLTDLNNHEAATAAIQEAIAAE GRVDVLVNNAGITRDATFKKMSYEQWSQVIDTNLKTLFT VTQPVFNKMLEQKSGRIVNISSVNGLKGQFGQANYSASKA GIIGFTKALAQEGARSNICVNVVAPGYTATPMVTAMREDV IKSIEAQIPLQRLAAPAEIAAAVMYLVSEHGAYVTGETLSIN GGLYMH
101	PhaC	Acinetoba cter sp.	MNPNSFQFKENILQFFSVHDDIWKKLQEFYYGQSPINEALA QLNKEDMSLFFEALSKNPARMMEMQWSWWQGQIQIYQN VLMRSVAKDVAPFIQPESGDRRFNSPLWQEHPNFDLLSQS YLLFSQLVQNMVDVVEGVPDKVRYRIHFFTRQMINALSPS NFLWTNPEVIQQTVAEQGENLVRGMQVFHDDVMNSGKY LSIRMVNSDSFSLGKDLAYTPGAVVFENDIFQLLQYEATTE NVYQTPILVVPPFINKYYVLDLREQNSLVNWLRQQGHTVF LMSWRNPNAEQKELTFADLITQGSVEALRVIEEITGEKEAN CIGYCIGGTLLAATQAYYVAKRLKNHVKSATYMATIIDFE NPGSLGVFINEPVVSGLENLNNQLGYFDGRQLAVTFSLLRE NTLYWNYYIDNYLKGKEPSDFDILYWNSDGTNIPAKIHNF LLRNLYLNNELISPNAVKVNGVGLNLSRVKTPSFFIATQED HIALWDTCFRGADYLGGESTLVLGESGHVAGIVNPPSRNK YGCYTNAAKFENTKQWLDGAEYHPESWWLRWQAWVTP YTGEQVPARNLGNAQYPSIEAAPGRYVLVNLF
102	phaABC	Acinetoba cter sp.	AAGCTTATAGCTAACACCGCAATCAATTTTTTCACTCGT CTAGCGTCTGTCAAGCGCGTATTTTCAAGATTAAACCCG CGTCCTTTGAGACAACTGAATAAGGTTTCAATTTCCCAG CGTAATGCATAATCCTGAATAGCATTGGCATTAAACTGA GGAGAAACGACGAGTAAAAGCTCTCCATTTTCTAACTGT AGTGCACTTATATATAGTTTCACCCGACCAACCAAAATC CGTCGTTTACGACATTCAATTTGACCAACTTTAAGATGG CGAAATAAATCACTAATTTTATGATTCTTTCCTAAATGA TTGGTGACAATGAAGTTTTTTTAACACGAATGCAGAAGT TGATGTCTTGGTTCAATTAACCATGTAAACCACTGCTCA CCGATAAACTCTCTGTCTGCGAACACATTCACAATACGG TCTTTACCAAAAATGGCTATAAAGCGTTGAATCAAAGCA ATACGCTCTTTCGTATCTGAATTTCCACGTTTATTAAGCA ATGTCCAAAGGATAGGTATCGCTATTCCACGATAAACG ATTGCGAGCATCAGGATATTAATATTTCGTTTTCCCCATT TCCAATTGGTTCTATCTAAAGTCAGTTGCACTTGGTCGA ATGAAAACATATTGAAAATCAACTGAGAAATTTGACGA TAATCAAAATACTGACCTGCAAAGAAGCGCTGCATACG TCGATAAAATGATTGTGGTAAGCACTTGATGGGCAAGG CTTTAGATGCAGAAGAAAGATTACATGTTTGCTTTAAAA TAATCACAAGCATGATGAGCGCAAAGCACTTTAAATGT GACTTGTTCCATTTTAGATATTTGTTTAAGATAAGATAT AACTCATTGAGATGTGTCATAGTATTCGTCGTTAGAAAA CAATTATTATGACATTATTTCAATGAGTTATCTATTTTTG TCGTGTACAGAGCAATATTTGTTTACTTTTGACTTTAAA GCATCATCAAACTGCGATCTGTTTGCAATATAAAACGCT TAATTTCTAAACAAGAATAAAGAGGAAAAACTTCTTATT TTTTTATAACCTTATTCTGCTTAGGAAAACAACATGTCT GAACAAAAAGTAGCTTTAGTGACGGGTGCATTAGGTGG TATTGGCAGTGAGATTTGCCGTCAACTGGTTACAGCAGG CTATAAAATTATCGCAACGGTTGTACCACGTGAAGAAG ACCGCGAAAAACAATGGCTTCAAAGTGAAGGATTTCAA GACAGCGATGTACGCTTTGTCTTAACGGATTTAAATAAC CACGAAGCGGCAACAGCAGCTATTCAAGAAGCAATTGC TGCTGAAGGTCGTGTCGATGTGCTGGTCAATAATGCAGG

-305WO 2017/023818

PCT/US2016/044922

CATCACCCGTGATGCAACTTTTAAAAAGATGAGCTATGA ACAGTGGTCACAAGTCATTGATACCAACTTAAAAACATT GTTCACAGTGACTCAGCCCGTGTTTAACAAAATGTTAGA ACAAAAGTCGGGACGTATTGTCAATATCAGCTCAGTCA ACGGTTTAAAAGGTCAGTTTGGACAAGCCAACTACTCTG CGAGCAAAGCCGGCATTATCGGTTTTACCAAAGCCTTAG CTCAAGAAGGTGCACGTTCAAATATTTGTGTAAACGTGG TTGCGCCTGGCTATACCGCAACACCAATGGTTACTGCCA TGCGTGAAGATGTGATTAAAAGCATTGAAGCACAAATT CCTCTACAACGTCTTGCTGCGCCAGCTGAAATTGCCGCT GCTGTTATGTACTTGGTCAGCGAGCACGGTGCGTACGTG ACAGGCGAAACCTTATCGATTAATGGCGGTTTATACATG CACTAAACCGTGCAGCCCCTATTTTCATTTACAAGTTTA TTTACTGGAGTTACACCATGCTATACGGCGACTTATTTT CAAATATGAATGCACAATACAAAAACGTATTTGAACCG TACACAAAATTCAACAGCTTAGTGGCTAAAAACTTTGCT GACTTAACCAACCTACAATTAGAAGCAGCACGCAACTA TGCCAACATTGGTCTAGCGCAAATGTTTGCCAATAGTGA AGTTAAAGACATGCAAAGCATGGTGAATTGCACCACCA AGCAATTAGAAACCATGAACAAACTTAGTCAGCAAATG ATTGAAGATGGCAAAAAGTTGGCAACACTAACGACTGA ATTCAAATCGGAATTTGAAAAGTTAGTTAGCGAATCTAT GCCTAACAATAAATAACACTGCTCTGAAAACCATGCGTT ATCAGGACGAATGTTACGGGGAAGTGTGAAAATTTCCC CGTTTTAGTTTCAGCCCTGCACTCAATTTGATTGCTAAA AGCCATGTGCTATGGAGCGATGAAATGAACCCGAACTC ATTTCAATTCAAAGAAAACATACTACAATTTTTTTCTGT ACATGATGACATCTGGAAAAAATTACAAGAATTTTATTA TGGGCAAAGCCCAATTAATGAGGCTTTGGCGCAGCTCA ACAAAGAAGATATGTCTTTGTTCTTTGAAGCACTATCTA AAAACCCAGCTCGCATGATGGAAATGCAATGGAGCTGG TGGCAAGGTCAAATACAAATCTACCAAAATGTGTTGAT GCGCAGCGTGGCCAAAGATGTAGCACCATTTATTCAGCC TGAAAGTGGTGATCGTCGTTTTAACAGCCCATTATGGCA AGAACACCCAAATTTTGACTTGTTGTCACAGTCTTATTT ACTGTTTAGCCAGTTAGTGCAAAACATGGTAGATGTGGT CGAAGGTGTTCCAGACAAAGTTCGCTATCGTATTCACTT CTTTACCCGCCAAATGATCAATGCGTTATCTCCAAGTAA CTTTCTGTGGACTAACCCAGAAGTGATTCAGCAAACTGT AGCTGAACAAGGTGAAAACTTAGTCCGTGGCATGCAAG TTTTCCATGATGATGTCATGAATAGCGGCAAGTATTTAT CTATTCGCATGGTGAATAGCGACTCTTTCAGCTTGGGCA AAGATTTAGCTTACACCCCTGGTGCAGTCGTCTTTGAAA ATGACATTTTCCAATTATTGCAATATGAAGCAACTACTG AAAATGTGTATCAAACCCCTATTCTAGTCGTACCACCGT TTATCAATAAATATTATGTGCTGGATTTACGCGAACAAA ACTCTTTAGTGAACTGGTTGCGCCAGCAAGGTCATACAG TCTTTTTAATGTCATGGCGTAACCCAAATGCCGAACAGA AAGAATTGACTTTTGCCGATCTCATTACACAAGGTTCAG TGGAAGCTTTGCGTGTAATTGAAGAAATTACCGGTGAA AAAGAGGCCAACTGCATTGGCTACTGTATTGGTGGTACG TTACTTGCTGCGACTCAAGCCTATTACGTGGCAAAACGC CTGAAAAATCACGTAAAGTCTGCGACCTATATGGCCACC ATTATCGACTTTGAAAACCCAGGCAGCTTAGGTGTATTT ATTAATGAACCTGTAGTGAGCGGTTTAGAAAACCTGAA CAATCAATTGGGTTATTTCGATGGTCGTCAGTTGGCAGT TACCTTCAGTTTACTGCGTGAAAATACGCTGTACTGGAA TTACTACATCGACAACTACTTAAAAGGTAAAGAACCTTC

-306WO 2017/023818

PCT/US2016/044922

			TGATTTTGATATTTTATATTGGAACAGCGATGGTACGAA TATCCCTGCCAAAATTCATAATTTCTTATTGCGCAATTTG TATTTGAACAATGAATTGATTTCACCAAATGCCGTTAAG GTTAACGGTGTGGGCTTGAATCTATCTCGTGTAAAAACA CCAAGCTTCTTTATTGCGACGCAGGAAGACCATATCGCA CTTTGGGATACTTGTTTCCGTGGCGCAGATTACTTGGGT GGTGAATCAACCTTGGTTTTAGGTGAATCTGGACACGTA GCAGGTATTGTCAATCCTCCAAGCCGTAATAAATACGGT TGCTACACCAATGCTGCCAAGTTTGAAAATACCAAACA ATGGCTAGATGGCGCAGAATATCACCCTGAATCTTGGTG GTTGCGCTGGCAGGCATGGGTCACACCGTACACTGGTG AACAAGTCCCTGCCCGCAACTTGGGTAATGCGCAGTATC CAAGCATTGAAGCGGCACCGGGTCGCTATGTTTTGGTAA ATTTATTCTAATCGGTCATATAACAACAGCCATGCAGAT GCTATATATCATGTGCATCCACAGAAACATGAACACAA AATTTAAGGATATAAAATGAAAGATGTTGTGATTGTTGC AGCAAAACGTACTGCGATTGGTAGCTTTTTAGGTAGTCT TGCATCTTTATCTGCACCACAGTTGGGGCAAACAGCAAT TCGTGCAGTTTTAGACAGCGCTAATGTAAAACCTGAACA AGTTGATCAGGTGATTATGGGCAACGTACTCACGACAG GCGTGGGACAAAACCCTGCACGTCAGGCAGCAATTGCT GCTGGTATTCCAGTACAAGTGCCTGCATCTACGCTGAAT GTCGTCTGTGGTTCAGGTTTGCGTGCGGTACATTTGGCA GCACAAGCCATTCAATGCGATGAAGCCGACATTGTGGT CGCAGGTGGTCAAGAATCTATGTCACAAAGTGCGCACT ATATGCAGCTGCGTAATGGGCAAAAAATGGGTAATGCA CAATTGGTGGATAGCATGGTGGCTGATGGTTTAACCGAT GCCTATAACCAGTATCAAATGGGTATTACCGCAGAAAA TATTGTAGAAAAACTGGGTTTAAACCGTGAAGAACAAG ATCAACTTGCATTGACTTCACAACAACGTGCTGCGGCAG CTCAGGCAGCTGGCAAGTTTAAAGATGAAATTGCCGTA GTCAGCATTCCACAACGTAAAGGTGAGCCTGTTGTATTT GCTGAAGATGAATACATTAAAGCCAATACCAGCCTTGA AAGCCTCACAAAACTACGCCCAGCCTTTAAAAAAGATG GTAGCGTAACCGCAGGTAATGCTTCAGGCATTAATGATG GTGCAGCAGCAGTACTGATGATGAGTGCGGACAAAGCA GCAGAATTAGGTCTTAAGCCATTGGCACGTATTAAAGGC TATGCCATGTCTGGTATTGAGCCTGAAATTATGGGGCTT GGTCCTGTCGATGCAGTAAAGAAAACCCTCAACAAAGC AGGCTGGAGCTTAGATCAGGTTGATTTGATTGAAGCCAA TGAAGCATTTGCTGCACAGGCTTTGGGTGTTGCTAAAGA ATTAGGCTTAGACCTGGATAAAGTCAACGTCAATGGCG GTGCAATTGCATTGGGTCACCCAATTGGGGCTTCAGGTT GCCGTATTTTGGTGACTTTATTACATGAAATGCAGCGCC GTGATGCCAAGAAAGGCATTGCAACCCTCTGTGTTGGCG GTGGTATGGGTGTTGCACTTGCAGTTGAACGTGACTAAG TACACCATTGCATCGAATCTTGAAACTTGATAAAGATTG ACAATAAATTCAATACATAATGGGAGCTCAGGCTTCCAT TATTTCTAGCTGAGCGCATTTCTAATATTAAGGCTTCTA GCTCAGCATTGATTTTAGTATTTGGCGATTTTAAGGGAC GTCTACTCTGACTACTTAATCCATCAATACCTTGCTCAG AATATCGTTTCCACCACTTGCGTAACGTTGGTCTAGA
103	AccA	E. coli	MSLNFLDFEQPIAELEAKIDSLTAVSRQDEKLDINIDEEVHR LREKSVELTRKIFADLGAWQIAQLARHPQRPYTLDYVRLA FDEFDELAGDRAYADDKAIVGGIARLDGRPVMIIGHQKGR ETKEKIRRNFGMPAPEGYRKALRLMQMAERFKMPIITFIDT PGAYPGVGAEERGQSEAIARNLREMSRLGVPVVCTVIGEG GSGGALAIGVGDKVNMLQYSTYSVISPEGCASILWKSADK

-307WO 2017/023818

PCT/US2016/044922

			APLAAEAMGIIAPRLKELKLIDSIIPEPLGGAHRNPEAMAAS LKAQLLADLADLDVLSTEDLKNRRYQRLMSYGYA
104	accA	E. coli	ATGAGTCTGAATTTCCTTGATTTTGAACAGCCGATTGCA GAGCTGGAAGCGAAAATCGATTCTCTGACTGCGGTTAG CCGTCAGGATGAGAAACTGGATATTAACATCGATGAAG AAGTGCATCGTCTGCGTGAAAAAAGCGTAGAACTGACA CGTAAAATCTTCGCCGATCTCGGTGCATGGCAGATTGCG CAACTGGCACGCCATCCACAGCGTCCTTATACCCTGGAT TACGTTCGCCTGGCATTTGATGAATTTGACGAACTGGCT GGCGACCGCGCGTATGCAGACGATAAAGCTATCGTCGG TGGTATCGCCCGTCTCGATGGTCGTCCGGTGATGATCAT TGGTCATCAAAAAGGTCGTGAAACCAAAGAAAAAATTC GCCGTAACTTTGGTATGCCAGCGCCAGAAGGTTACCGCA AAGCACTGCGTCTGATGCAAATGGCTGAACGCTTTAAG ATGCCTATCATCACCTTTATCGACACCCCGGGGGCTTAT CCTGGCGTGGGCGCAGAAGAGCGTGGTCAGTCTGAAGC CATTGCACGCAACCTGCGTGAAATGTCTCGCCTCGGCGT ACCGGTAGTTTGTACGGTTATCGGTGAAGGTGGTTCTGG CGGTGCGCTGGCGATTGGCGTGGGCGATAAAGTGAATA TGCTGCAATACAGCACCTATTCCGTTATCTCGCCGGAAG GTTGTGCGTCCATTCTGTGGAAGAGCGCCGACAAAGCG CCGCTGGCGGCTGAAGCGATGGGTATCATTGCTCCGCGT CTGAAAGAACTGAAACTGATCGACTCCATCATCCCGGA ACCACTGGGTGGTGCTCACCGTAACCCGGAAGCGATGG CGGCATCGTTGAAAGCGCAACTGCTGGCGGATCTGGCC GATCTCGACGTGTTAAGCACTGAAGATTTAAAAAATCGT CGTTATCAGCGCCTGATGAGCTACGGTTACGCGTAA
105	MmcE	Pelotomacul um thermopropi onicum	MFKQDQLDKIAAKKESWSAKLAAAVKKRPEREAQFMTDS GIEVNTVYTPLDIADMDYERDLGLPGEYPYTRGVQPNMYR GRLWTMRQYAGFGTAEETNQRFRYLLEQGQTGLSCAFDL PTQIGYDSDHPMARGEIGKVGVAIDSLQDMETLFDQIPLGK VSTSMTINAPAGILLAMYIVVAEKQGFKRAELNGTIQNDII KEYVGRGTYILPPEPSMRLITNIFEFCSKEVPNWNTISISGYH IREAGCTAAQEIAFTLADGIAYVDAAIKAGLDVDQFGPRLS FFFNAHLNFLEEIAKFRAARRVWAKIMKERFGAKDPRSWT LRFHTQTAGCSLTAQQPMVNIMRTAFEALAAVLGGTQSLH TNSYDEALALPSDESVLIALRTQQVIGYEIGVCDVVDPLGG SYYIESLTNQLEAKAWEYIEKIDALGGAVKAIDYMQKEIH NAAYQYQLAIDNKKKTVIGVNKFQLKEEEKPKNLLKVDLS VGERQIAKLKKLKEERDNAKVEALLKQVREAAQSDANM MPVFIDAVKEYVTLGEICGVLRDVFGEYKQQIVF
106	mmcE	Pelotomacul um thermopropi onicum	TTGTTTAAACAGGATCAACTGGACAAAATTGCTGCCAAG AAAGAAAGCTGGTCTGCAAAGCTGGCAGCAGCGGTCAA AAAGCGTCCGGAAAGAGAAGCTCAATTCATGACCGACT CTGGAATTGAAGTCAACACCGTTTACACTCCTCTTGATA TTGCAGACATGGATTATGAGCGTGACCTGGGCCTGCCTG GGGAATACCCGTATACCCGGGGTGTGCAGCCTAACATG TACCGCGGCCGCCTCTGGACCATGCGCCAGTACGCAGGT TTTGGCACAGCCGAAGAAACCAACCAGCGTTTCCGCTAT CTCCTGGAGCAAGGGCAGACAGGCCTTAGCTGCGCCTTC GATTTGCCTACTCAGATCGGCTACGATTCGGACCATCCT ATGGCAAGGGGAGAAATCGGTAAGGTTGGCGTTGCTAT AGACTCCCTGCAGGACATGGAAACTCTTTTCGACCAGAT CCCCCTGGGCAAGGTCAGCACTTCCATGACCATCAACGC

-308WO 2017/023818

PCT/US2016/044922

			CCCGGCAGGCATACTACTGGCCATGTATATTGTGGTGGC TGAAAAACAGGGGTTTAAGAGGGCAGAATTAAACGGAA CGATTCAAAACGATATTATTAAGGAATATGTCGGCCGG GGAACATACATCCTGCCGCCTGAGCCCTCAATGCGTTTA ATTACAAATATTTTTGAGTTCTGTTCCAAAGAAGTGCCC AACTGGAATACGATCAGCATCAGCGGCTATCATATCCGT GAAGCGGGTTGCACCGCAGCTCAGGAAATAGCCTTTAC CCTAGCGGACGGCATTGCCTATGTGGATGCAGCCATTAA AGCAGGCCTGGATGTTGATCAGTTTGGTCCTCGCCTTTC ATTCTTCTTCAATGCTCACCTGAACTTCCTCGAGGAAAT TGCAAAATTCCGGGCGGCACGGCGCGTCTGGGCGAAGA TTATGAAGGAACGTTTCGGAGCCAAAGATCCGCGCTCGT GGACCCTGCGCTTCCACACTCAGACTGCCGGCTGCAGCC TGACGGCCCAGCAGCCGATGGTAAATATCATGAGGACC GCATTTGAGGCCCTGGCTGCCGTACTGGGCGGGACTCAG TCCCTGCACACCAACTCCTATGACGAAGCCCTGGCCCTT CCCAGCGACGAGTCGGTGCTTATTGCATTGCGCACACAG CAGGTGATCGGCTATGAAATCGGCGTTTGCGACGTGGTT GACCCGCTTGGCGGATCCTACTACATTGAAAGCCTGACC AACCAGCTTGAAGCAAAAGCCTGGGAGTACATTGAGAA GATTGATGCCCTCGGCGGTGCCGTAAAGGCCATCGATTA CATGCAGAAGGAGATCCACAACGCCGCTTACCAGTATC AACTGGCTATTGACAATAAGAAGAAGACCGTTATCGGA GTGAACAAATTCCAGTTGAAGGAAGAAGAAAAGCCAAA GAACCTGCTGAAAGTGGACCTCTCCGTGGGCGAACGGC AGATTGCGAAGCTCAAAAAGCTTAAGGAAGAAAGAGAT AACGCCAAGGTTGAAGCCCTGCTGAAACAAGTGCGCGA GGCGGCGCAGAGCGATGCAAACATGATGCCTGTCTTTAT CGATGCGGTTAAGGAATACGTTACTCTGGGCGAGATCTG CGGCGTCCTGAGAGACGTATTCGGCGAATACAAGCAGC AAATCGTATTCTAG
107	Acs	E. coli	MSQIHKHTIPANIADRCLINPQQYEAMYQQSINVPDTFWGE QGKILDWIKPYQKVKNTSFAPGNVSIKWYEDGTLNLAANC LDRHLQENGDRTAIIWEGDDASQSKHISYKELHRDVCRFA NTLLELGIKKGDVVAIYMPMVPEAAVAMLACARIGAVHS VIFGGFSPEAVAGRIIDSNSRLVITSDEGVRAGRSIPLKKNV DDALKNPNVTSVEHVVVLKRTGGKIDWQEGRDLWWHDL VEQASDQHQAEEMNAEDPLFILYTSGSTGKPKGVLHTTGG YLVYAALTFKYVFDYHPGDIYWCTADVGWVTGHSYLLY GPLACGATTLMFEGVPNWPTPARMAQVVDKHQVNILYTA PTAIRALMAEGDKAIEGTDRSSLRILGSVGEPINPEAWEWY WKKIGNEKCPVVDTWWQTETGGFMITPLPGATELKAGSA TRPFFGVQPALVDNEGNPLEGATEGSLVITDSWPGQARTLF GDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITGRVD DVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIPHNIKGQA IYAYVTLNHGEEPSPELYAEVRNWVRKEIGPLATPDVLHW TDSLPKTRSGKIMRRILRKIAAGDTSNLGDTSTLADPGVVE KLLEEKQAIAMPS
108	acs	E. coli	ATGAGCCAAATTCACAAACACACCATTCCTGCCAACATC GCAGACCGTTGCCTGATAAACCCTCAGCAGTACGAGGC GATGTATCAACAATCTATTAACGTACCTGATACCTTCTG GGGCGAACAGGGAAAAATTCTTGACTGGATCAAACCTT ACCAGAAGGTGAAAAACACCTCCTTTGCCCCCGGTAAT GTGTCCATTAAATGGTACGAGGACGGCACGCTGAATCT

-309WO 2017/023818

PCT/US2016/044922

			GGCGGCAAACTGCCTTGACCGCCATCTGCAAGAAAACG GCGATCGTACCGCCATCATCTGGGAAGGCGACGACGCC AGCCAGAGCAAACATATCAGCTATAAAGAGCTGCACCG CGACGTCTGCCGCTTCGCCAATACCCTGCTCGAGCTGGG CATTAAAAAAGGTGATGTGGTGGCGATTTATATGCCGAT GGTGCCGGAAGCCGCGGTTGCGATGCTGGCCTGCGCCC GCATTGGCGCGGTGCATTCGGTGATTTTCGGCGGCTTCT CGCCGGAAGCCGTTGCCGGGCGCATTATTGATTCCAACT CACGACTGGTGATCACTTCCGACGAAGGTGTGCGTGCCG GGCGCAGTATTCCGCTGAAGAAAAACGTTGATGACGCG CTGAAAAACCCGAACGTCACCAGCGTAGAGCATGTGGT GGTACTGAAGCGTACTGGCGGGAAAATTGACTGGCAGG AAGGGCGCGACCTGTGGTGGCACGACCTGGTTGAGCAA GCGAGCGATCAGCACCAGGCGGAAGAGATGAACGCCGA AGATCCGCTGTTTATTCTCTACACCTCCGGTTCTACCGGT AAGCCAAAAGGTGTGCTGCATACTACCGGCGGTTATCTG GTGTACGCGGCGCTGACCTTTAAATATGTCTTTGATTAT CATCCGGGTGATATCTACTGGTGCACCGCCGATGTGGGC TGGGTGACCGGACACAGTTACTTGCTGTACGGCCCGCTG GCCTGCGGTGCGACCACGCTGATGTTTGAAGGCGTACCC AACTGGCCGACGCCTGCCCGTATGGCGCAGGTGGTGGA CAAGCATCAGGTCAATATTCTCTATACCGCACCCACGGC GATCCGCGCGCTGATGGCGGAAGGCGATAAAGCGATCG AAGGCACCGACCGTTCGTCGCTGCGCATTCTCGGTTCCG TGGGCGAGCCAATTAACCCGGAAGCGTGGGAGTGGTAC TGGAAAAAAATCGGCAACGAGAAATGTCCGGTGGTCGA TACCTGGTGGCAGACCGAAACCGGCGGTTTCATGATCAC CCCGCTGCCTGGCGCTACCGAGCTGAAAGCCGGTTCGGC AACACGTCCGTTCTTCGGCGTGCAACCGGCGCTGGTCGA TAACGAAGGTAACCCGCTGGAGGGGGCCACCGAAGGTA GCCTGGTAATCACCGACTCCTGGCCGGGTCAGGCGCGTA CGCTGTTTGGCGATCACGAACGTTTTGAACAGACCTACT TCTCCACCTTCAAAAATATGTATTTCAGCGGCGACGGCG CGCGTCGCGATGAAGATGGCTATTACTGGATAACCGGG CGTGTGGACGACGTGCTGAACGTCTCCGGTCACCGTCTG GGGACGGCAGAGATTGAGTCGGCGCTGGTGGCGCATCC GAAGATTGCCGAAGCCGCCGTAGTAGGTATTCCGCACA ATATTAAAGGTCAGGCGATCTACGCCTACGTCACGCTTA ATCACGGGGAGGAACCGTCACCAGAACTGTACGCAGAA GTCCGCAACTGGGTGCGTAAAGAGATTGGCCCGCTGGC GACGCCAGACGTGCTGCACTGGACCGACTCCCTGCCTAA AACCCGCTCCGGCAAAATTATGCGCCGTATTCTGCGCAA AATTGCGGCGGGCGATACCAGCAACCTGGGCGATACCT CGACGCTTGCCGATCCTGGCGTAGTCGAGAAGCTGCTTG AAGAGAAGCAGGCTATCGCGATGCCATCGTAA
109	MutA	Propionibac terium freudenreich ii subsp. shermanii	MSSTDQGTNPADTDDLTPTTLSLAGDFPKATEEQWEREVE KVLNRGRPPEKQLTFAECLKRLTVHTVDGIDIVPMYRPKD APKKLGYPGVAPFTRGTTVRNGDMDAWDVRALHEDPDE KFTRKAILEGLERGVTSLLLRVDPDAIAPEHLDEVLSDVLL EMTKVEVFSRYDQGAAAEALVSVYERSDKPAKDLALNLG LDPIAFAALQGTEPDLTVLGDWVRRLAKFSPDSRAVTIDA NIYHNAGAGDVAELAWALATGAEYVRALVEQGFTATEAF DTINFRVTATHDQFLTIARLRALREAWARIGEVFGVDEDK RGARQNAITSWRDVTREDPYVNILRGSIATFSASVGGAESI

-310WO 2017/023818

PCT/US2016/044922

			TTLPFTQALGLPEDDFPLRIARNTGIVLAEEVNIGRVNDPAG GSYYVESLTRSLADAAWKEFQEVEKLGGMSKAVMTEHVT KVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFP AAPARKGLAWHRDSEVFEQLMDRSTSVSERPKVFLACLGT RRDFGGREGFSSPVWHIAGIDTPQVEGGTTAEIVEAFKKSG AQVADLCSSAKVYAQQGLEVAKALKAAGAKALYLSGAF KEFGDDAAEAEKLIDGRLFMGMDVVDTLSSTLDILGVAK
110	muta	Propionibac terium freudenreich ii subsp. shermanii	TCAGGCCTCCAGCTTGTCCAGGGTGGAGGTCAGCAACTC CACCACATTCATTCCGTCGAAGACGTTGCCGTCGATCAC GGCGTTCACCTCGGCCTCGTCGCCGCCGAGTTCCTTCAG CTGCCCGGCGAGCCGCACCTCCTGGGCGCCCGCCTCCTT CAGGGCCTTCGCGACGGCGAGACCGTGGGCGGCGTAGA CCTTCGCGCTGGAGCACAGGACCGCGATGTCGGTGCCC GCCTCCTGCATGGCCTTGACGAACACCTCCGGGTTGGTG CCCTCCGCGATCACGGTGTTGATGCCACCCACGTGGTAC AGGTTCGAGGTGAAGCCCTCGCGACCACCGAAGTCGCG CCGGGTGCCGAGGCAGGCCAGCAGCACGGTCGGGGTCT TCTTGGCGGCCTTGGAACGGTCCCGGAGGTCCTCGAAGA CCTGGCTGTCGCGCACGAACGGGATGCCGCCGAGCTTC GGGGCCGCGGGGCGCGGGGCGCGTTCGAGGGCCTTCTC GAGGTGGTTCGGGAACATCGAGACGCCCGTCAGCGGCA GCTTGCGGGTGGCGAGCAGCTTGGCGCGGGCCTCGTTG ATTTCCTTGAGCTGGGCGGCCACGGTGCCATCGGCGATG GCGGCAGCCATACCCTTCTCGTCGAGCTGACCGAACAGC TCCCAGGCCTTCTCGCAGAGCTGCTTGGTCATGGACTCG ACGAACCATGCGCCGCCGGCCGGGTCGTTGACGCGGCC GATGTTCGACTCCTCGGCCAGCACCACCTGGGTGTTGCG GGCGATACGGCGGGTCAGGACGTCGGGAAGACCGATCA CGGTGTCGAGCGGCAACACGGTGATGAACTCGGCCTGG CCGACGGCCGCGGCGAAGGCCGAGATGGTGCCGCGCAG CACGTTGACGTAGGCGTCGTCGCGGGTGATCTCGCGCAG CGACGTGACGGCGTGCTGCACCGCGCCGCGTTTCTCCGG GCTCACCCCGAGCACCTCGCCGACCCGGTTCCACAGGGT GCGCAGCGCGCGCAGCCGGGAGATGGTGATGAACTGGT TGGTGTTCGCGGAGACCCGGAACAGGATGCTGTCGAAG GCCTCGTCGGCGCTCAGCCCGAGATCGGTGAGGGCGCG CACGTACTCGATGCCCGTGGCCAGCGCGTAGGCCAGCT GGGCGACGTCACCGGCGCCCATGGAATCGTAACGGGAG GCGTCCACGACGATGGGACGCACGCCCGAGAACGGCTT TGCAAGCTCCACGGCCCTCGCGATCACCGAGAGGTCGG GGGTGGTTCCGTTGAGGGCGGCGAAACCGATCGGGTCG ATGCCGAGGCTGCCGCGGATGTTCTCCTTACCGGAGGCG GCGAAAGCCGCGGCCAGGGCCTCGGCGGCCGCCAGCTC ATCGGTGTTGGAGGAAACATGTGTGGGGGCAAGGTCGA ACAGGACATCGCTCAGGACCTCAGCCAGCTTGTCTGCGG GGACCGCATCGGGATCCACGCGCACCCAGACCGCGGAG GTGCCGCGCTCCAGGTCGGTGTCCACGGCCTTGCGGGCC TCGGCCGGGTCGGGTTCCTCAATGAGCTGAGCACTGAAC CAGCCCTCATCCATCTCTCCTGCACGGACCGTGGTGCCG CGCGTGAATGGGGCCACTCCGGGGAAACCAAGCTCCTT GACGCCATCGTCAATGGTGTACAGCGGCTTGATCACAA GCCCATCGACGGTGTGGCTCGTCAGGCGCTTGTATGCCT GCTCAATGTTGAGTTCCTTGCCCTCGGGACGCCTCCGGT TCAGTACCTTCAGCACCTCTTTCTCCCAGTCTGCAAGGC

-311WO 2017/023818

PCT/US2016/044922

			TGGGAGTGGCGAAGTCAGCGGCGAGACTGATCTCGGCC GCGCTCGTTGATTCTGCGCTCAT
111	MutB	Propionibac terium freudenreich ii subsp. shermanii	MSTLPRFDSVDLGNAPVPADAARRFEELAAKAGTGEAWE TAEQIPVGTLFNEDVYKDMDWLDTYAGIPPFVHGPYATM YAFRPWTIRQYAGFSTAKESNAFYRRNLAAGQKGLSVAFD LPTHRGYDSDNPRVAGDVGMAGVAIDSIYDMRELFAGIPL DQMSVSMTMNGAVLPILALYVVTAEEQGVKPEQLAGTIQ NDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPKWNSISI SGYHMQEAGATADIEMAYTLADGVDYIRAGESVGLNVDQ FAPRLSFFWGIGMNFFMEVAKLRAARMLWAKLVHQFGPK NPKSMSLRTHSQTSGWSLTAQDVYNNVVRTCIEAMAATQ GHTQSLHTNSLDEAIALPTDFSARIARNTQLFLQQESGTTR VIDPWSGSAYVEELTWDLARKAWGHIQEVEKVGGMAKAI EKGIPKMRIEEAAARTQARIDSGRQPLIGVNKYRLEHEPPL DVLKVDNSTVLAEQKAKLVKLRAERDPEKVKAALDKITW AAGNPDDKDPDRNLLKLCIDAGRAMATVGEMSDALEKVF GRYTAQIRTISGVYSKEVKNTPEVEEARELVEEFEQAEGRR PRILLAKMGQDGHDRGQKVIATAYADLGFDVDVGPLFQTP EETARQAVEADVHVVGVSSLAGGHLTLVPALRKELDKLG RPDILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLV KKLRASLDA
112	mutab	Propionibac terium freudenreich ii subsp. shermanii	GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTCGGC AATGCCCCGGTTCCTGCTGATGCCGCACGACGCTTCGAG GAACTGGCCGCCAAGGCCGGCACCGGAGAGGCGTGGGA GACGGCCGAGCAGATTCCGGTTGGCACCCTGTTCAACG AAGACGTCTACAAGGACATGGACTGGCTGGACACCTAC GCAGGTATCCCGCCGTTCGTCCACGGCCCGTATGCAACC ATGTACGCGTTCCGTCCCTGGACGATTCGCCAGTACGCC GGTTTCTCCACGGCCAAGGAGTCGAACGCCTTCTACCGC CGCAACCTTGCGGCCGGCCAGAAGGGCCTGTCGGTTGC CTTCGACCTGCCCACCCACCGTGGCTACGACTCGGACAA TCCCCGCGTCGCCGGTGACGTCGGCATGGCCGGTGTGGC CATCGACTCCATCTATGACATGCGCGAGCTGTTCGCCGG CATTCCGCTGGACCAGATGAGCGTGTCCATGACCATGAA CGGCGCCGTGCTGCCGATCCTGGCCCTCTATGTGGTGAC CGCCGAGGAGCAGGGCGTCAAGCCCGAGCAGCTCGCCG GGACGATCCAGAACGACATCCTCAAGGAGTTCATGGTT CGTAACACCTACATCTACCCGCCGCAGCCGAGTATGCGA ATCATCTCTGAGATCTTCGCCTACACGAGTGCCAATATG CCGAAGTGGAATTCGATTTCCATTTCCGGCTACCACATG CAGGAAGCCGGCGCCACGGCCGACATCGAGATGGCCTA TACCCTGGCCGACGGTGTTGACTACATCCGCGCCGGCGA GTCGGTGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCT GTCCTTCTTCTGGGGCATCGGCATGAACTTCTTCATGGA GGTTGCCAAGCTGCGTGCCGCGCGCATGTTGTGGGCCAA GCTGGTGCATCAGTTCGGGCCGAAGAACCCGAAGTCGA TGAGCCTGCGCACCCACTCGCAGACCTCCGGTTGGTCGC TGACCGCCCAGGACGTCTACAACAACGTCGTGCGTACCT GCATCGAGGCCATGGCCGCCACCCAGGGCCATACCCAG TCGCTGCACACGAACTCGCTCGACGAGGCCATCGCCCTG CCGACCGATTTCAGCGCCCGCATCGCCCGTAACACCCAG CTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTGAT CGACCCGTGGAGCGGCTCGGCATACGTCGAGGAGCTCA CCTGGGACCTGGCCCGCAAGGCATGGGGTCACATCCAG GAGGTCGAGAAGGTCGGCGGCATGGCCAAGGCCATCGA

-312WO 2017/023818

PCT/US2016/044922

			AAAGGGCATCCCCAAGATGCGCATCGAGGAAGCCGCCG CCCGCACCCAGGCACGCATCGACTCCGGCCGCCAGCCG CTGATCGGCGTGAACAAGTACCGCCTGGAGCACGAGCC GCCGCTCGATGTGCTCAAGGTGGACAACTCCACGGTGCT CGCCGAGCAGAAGGCCAAGCTGGTCAAGCTGCGCGCCG AGCGCGATCCCGAGAAGGTCAAGGCCGCCCTCGACAAG ATCACCTGGGCCGCCGGCAACCCCGACGACAAGGATCC GGATCGCAACCTGCTGAAGCTGTGCATCGACGCTGGCC GCGCCATGGCGACGGTCGGCGAGATGAGCGACGCGCTC GAGAAGGTCTTCGGACGCTACACCGCCCAGATTCGCAC CATCTCCGGTGTGTACTCGAAGGAAGTGAAGAACACGC CTGAGGTTGAGGAAGCACGCGAGCTCGTTGAGGAATTC GAGCAGGCCGAGGGCCGTCGTCCTCGCATCCTGCTGGCC AAGATGGGCCAGGACGGTCACGACCGTGGCCAGAAGGT CATCGCCACCGCCTATGCCGACCTCGGTTTCGACGTCGA CGTGGGCCCGCTGTTCCAGACCCCGGAGGAGACCGCAC GTCAGGCCGTCGAGGCCGATGTGCACGTGGTGGGCGTTT CGTCGCTCGCCGGCGGGCATCTGACGCTGGTTCCGGCCC TGCGCAAGGAGCTGGACAAGCTCGGACGTCCCGACATC CTCATCACCGTGGGCGGCGTGATCCCTGAGCAGGACTTC GACGAGCTGCGTAAGGACGGCGCCGTGGAGATCTACAC CCCCGGCACCGTCATTCCGGAGTCGGCGATCTCGCTGGT CAAGAAACTGCGGGCTTCGCTCGATGCCTAG
113	CobB	E. coli	MEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVAT PEGFDRDPELVQAFYNARRRQLQQPEIQPNAAHLALAKLQ DALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQ SGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMPLG MDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHT VELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAG SIA
114	cobB	E. coli	ATGGAAAAACCAAGAGTACTCGTACTGACAGGGGCAGG AATTTCTGCGGAATCAGGTATTCGTACCTTTCGCGCCGC AGATGGCCTGTGGGAAGAACATCGGGTTGAAGATGTGG CAACGCCGGAAGGTTTCGATCGCGATCCTGAACTGGTGC AAGCGTTTTATAACGCCCGTCGTCGACAGCTGCAGCAGC CAGAAATTCAGCCTAACGCCGCGCATCTTGCGCTGGCTA AACTGCAAGACGCCCTCGGCGATCGCTTTTTGCTGGTGA CGCAGAATATCGACAACCTGCATGAACGCGCAGGTAAT ACCAATGTGATTCATATGCATGGGGAACTGCTGAAAGT GCGTTGTTCACAAAGTGGTCAGGTTCTCGACTGGACAGG AGACGTTACCCCAGAAGATAAATGCCATTGTTGCCAGTT TCCGGCACCCTTGCGCCCGCACGTAGTGTGGTTTGGCGA AATGCCACTCGGCATGGATGAAATTTATATGGCGTTGTC GATGGCCGATATTTTCATTGCCATTGGCACATCCGGGCA TGTTTATCCGGCGGCTGGGTTTGTTCACGAAGCGAAACT GCATGGCGCGCACACCGTGGAACTGAATCTTGAACCGA GTCAGGTTGGTAATGAATTTGCCGAGAAATATTACGGCC CGGCAAGCCAGGTGGTGCCTGAGTTTGTTGAAAAGTTGC TGAAGGGATTAAAAGCGGGAAGCATTGCCTGA
115	Pka	E. coli	MSQRGLEALLRPKSIAVIGASMKPNRAGYLMMRNLLAGG FNGPVLPVTPAWKAVLGVLAWPDIASLPFTPDLAVLCTNA SRNLALLEELGEKGCKTCIILSAPASQHEDLRACALRHNMR LLGPNSLGLLAPWQGLNASFSPVPIKRGKLAFISQSAAVSN TILDWAQQRKMGFSYFIALGDSLDIDVDELLDYLARDSKT

-313WO 2017/023818

PCT/US2016/044922

			SAILLYLEQLSDARRFVSAARSASRNKPILVIKSGRSPAAQR LLNTTAGMDPAWDAAIQRAGLLRVQDTHELFSAVETLSH MRPLRGDRLMIISNGAAPAALALDALWSRNGKLATLSEET CQKLRDALPEHVAISNPLDLRDDASSEHYIKTLDILLHSQD FDALMVIHSPSAAAPATESAQVLIEAVKHHPRSKYVSLLTN WCGEHSSQEARRLFSEAGLPTYRTPEGTITAFMHMVEYRR NQKQLRETPALPSNLTSNTAEAHLLLQQAIAEGATSLDTHE VQPILQAYGMNTLPTWIASDSTEAVHIAEQIGYPVALKLRS PDIPHKSEVQGVMLYLRTANEVQQAANAIFDRVKMAWPQ ARVHGLLVQSMANRAGAQELRVVVEHDPVFGPLIMLGEG GVEWRPEDQAVVALPPLNMNLARYLVIQGIKSKKIRARSA LRPLDVAGLSQLLVQVSNLIVDCPEIQRLDIHPLLASGSEFT ALDVTLDISPFEGDNESRLAVRPYPHQLEEWVELKNGERC LFRPILPEDEPQLQQFISRVTKEDLYYRYFSEINEFTHEDLA NMTQIDYDREMAFVAVRRIDQTEEILGVTRAISDPDNIDAE FAVLVRSDLKGLGLGRRLMEKLITYTRDHGLQRLNGITMP NNRGMVALARKLGFNVDIQLEEGIVGLTLNLAQREES
116	pka	E. coli	ATGAGTCAGCGAGGACTGGAAGCACTACTGCGACCAAA ATCGATAGCGGTAATTGGCGCGTCGATGAAACCCAATC GCGCAGGTTACCTGATGATGCGTAACCTGCTGGCGGGA GGCTTTAACGGACCGGTACTCCCGGTGACGCCAGCCTGG AAAGCGGTGTTGGGTGTGTTGGCCTGGCCGGATATTGCC AGCTTGCCCTTTACACCCGACCTTGCGGTTTTATGTACC AATGCCAGCCGTAATCTTGCTCTTCTGGAAGAGCTCGGC GAGAAAGGCTGTAAAACCTGCATTATTCTTTCCGCCCCG GCATCGCAACACGAAGATCTCCGCGCCTGCGCCCTGCGC CATAACATGCGCCTGCTTGGACCAAACAGTCTGGGTTTA CTGGCTCCCTGGCAAGGTCTGAATGCCAGCTTTTCGCCT GTGCCGATTAAACGCGGCAAGCTGGCGTTTATTTCGCAA TCGGCTGCCGTCTCCAACACCATCCTCGACTGGGCGCAA CAGCGTAAGATGGGCTTTTCCTACTTTATTGCGCTCGGC GACAGCCTGGATATCGACGTTGATGAATTGCTTGACTAT CTGGCACGCGACAGTAAAACCAGCGCCATCCTGCTCTAT CTCGAACAGTTAAGCGACGCGCGACGCTTTGTTTCGGCG GCCCGTAGTGCCTCGCGTAATAAACCGATTCTGGTGATT AAAAGCGGACGTAGCCCGGCGGCACAGCGACTGCTCAA CACGACGGCAGGAATGGACCCGGCATGGGATGCGGCTA TTCAGCGTGCCGGTTTGTTGCGGGTACAGGACACCCACG AGCTGTTTTCGGCGGTGGAAACCCTTAGCCATATGCGCC CGCTACGTGGCGACCGGCTGATGATTATCAGCAACGGT GCTGCGCCTGCCGCGCTGGCGCTGGATGCCTTATGGTCA CGCAATGGCAAGCTGGCAACGCTAAGCGAAGAAACCTG CCAGAAACTGCGCGATGCACTGCCAGAACATGTGGCAA TATCTAACCCGCTCGATCTACGCGATGACGCCAGCAGTG AGCACTATATTAAAACGCTGGATATTCTGCTCCACAGCC AGGATTTTGACGCGCTGATGGTTATTCATTCGCCCAGCG CCGCTGCTCCCGCAACAGAAAGCGCGCAAGTATTAATT GAAGCGGTAAAGCATCATCCCCGCAGCAAATATGTCTCT TTGCTGACGAACTGGTGCGGCGAGCACTCCTCGCAAGA GGCACGACGTTTATTCAGCGAAGCCGGGCTGCCGACCT ACCGTACCCCGGAAGGAACCATCACTGCTTTTATGCATA TGGTGGAGTACCGGCGTAATCAGAAGCAACTACGCGAA ACGCCGGCGTTGCCCAGCAATCTGACTTCCAATACCGCA GAAGCGCATCTTCTGTTGCAACAGGCGATTGCCGAAGG

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PCT/US2016/044922

			GGCTACGTCGCTCGATACCCATGAAGTTCAGCCCATCCT GCAAGCGTATGGCATGAACACGCTCCCTACCTGGATTGC CAGCGATAGCACCGAAGCGGTGCATATTGCCGAACAGA TTGGTTATCCGGTGGCGCTGAAATTGCGTTCGCCGGATA TTCCACATAAATCGGAAGTTCAGGGCGTCATGCTTTACC TGCGTACAGCCAATGAAGTCCAGCAAGCGGCGAACGCT ATTTTCGATCGCGTAAAAATGGCCTGGCCACAGGCGCG GGTCCACGGCCTGTTGGTGCAAAGTATGGCTAACCGTGC TGGCGCTCAGGAGTTGCGGGTTGTGGTTGAGCACGATCC GGTTTTCGGGCCGTTGATCATGCTGGGTGAAGGCGGTGT GGAGTGGCGTCCTGAAGATCAAGCCGTCGTCGCACTGC CGCCGCTGAACATGAACCTGGCCCGCTATCTGGTTATTC AGGGGATCAAAAGTAAAAAGATTCGTGCGCGCAGTGCG CTACGCCCATTGGATGTTGCAGGCTTGAGCCAGCTTCTG GTGCAGGTTTCCAACTTGATTGTCGATTGCCCGGAAATT CAGCGTCTGGATATTCATCCTTTGCTGGCTTCTGGCAGT GAATTTACCGCGCTGGATGTCACGCTGGATATCTCGCCG TTTGAAGGCGATAACGAGAGTCGGCTGGCAGTGCGCCC TTATCCGCATCAGCTGGAAGAATGGGTAGAATTGAAAA ACGGTGAACGCTGCTTGTTCCGCCCGATTTTGCCAGAAG ATGAGCCACAACTTCAGCAATTCATTTCGCGAGTCACCA AAGAAGATCTTTATTACCGCTACTTTAGCGAGATCAACG AATTTACCCATGAAGATTTAGCCAACATGACACAGATCG ACTACGATCGGGAAATGGCGTTTGTAGCGGTACGACGT ATTGATCAAACGGAAGAGATCCTCGGCGTCACGCGTGC GATTTCCGATCCTGATAACATCGATGCCGAATTTGCTGT ACTGGTTCGCTCGGATCTCAAAGGGTTAGGCTTAGGTCG ACGCTTAATGGAAAAGTTGATTACCTATACGCGAGATCA CGGACTACAACGTCTGAATGGTATTACGATGCCAAACA ATCGTGGCATGGTGGCGCTAGCCCGCAAGCTCGGGTTTA ACGTTGATATCCAGCTCGAAGAGGGGATCGTTGGGCTTA CGCTAAATCTTGCCCAGCGCGAGGAATCATGA
117	DcuC	E. coli	MLTFIELLIGVVVIVGVARYIIKGYSATGVLFVGGLLLLIISA IMGHKVLPSSQASTGYSATDIVEYVKILLMSRGGDLGMMI MMLCGFAAYMTHIGANDMVVKLASKPLQYINSPYLLMIA AYFVACLMSLAVSSATGLGVLLMATLFPVMVNVGISRGA AAAICASPAAIILAPTSGDVVLAAQASEMSLIDFAFKTTLPIS IAAIIGMAIAHFFWQRYLDKKEHISHEMLDVSEITTTAPAFY AILPFTPIIGVLIFDGKWGPQLHIITILVICMLIASILEFLRSFN TQKVFSGLEVAYRGMADAFANVVMLLVAAGVFAQGLSTI GFIQSLISIATSFGSASIILMLVLVILTMLAAVTTGSGNAPFY AFVEMIPKLAHSSGINPAYLTIPMLQASNLGRTLSPVSGVV VAVAGMAKISPFEVVKRTSVPVLVGLVIVIVATELMVPGT AAAVTGK
118	dcuc	E. coli	ATGCTGACATTCATTGAGCTCCTTATTGGGGTTGTGGTT ATTGTGGGTGTAGCTCGCTACATCATTAAAGGGTATTCC GCCACTGGTGTGTTATTTGTCGGTGGCCTGTTATTGCTG ATTATCAGTGCCATTATGGGGCACAAAGTGTTACCGTCC AGCCAGGCTTCAACAGGCTACAGCGCCACGGATATCGT TGAATACGTTAAAATATTACTAATGAGCCGCGGCGGCG ACCTCGGCATGATGATTATGATGCTGTGTGGATTTGCCG CTTACATGACCCATATCGGCGCGAATGATATGGTGGTCA AGCTGGCGTCAAAACCATTGCAGTATATTAACTCCCCTT ACCTGCTGATGATTGCCGCCTATTTTGTCGCCTGTCTGAT

-315WO 2017/023818

PCT/US2016/044922

			GTCTCTGGCCGTCTCTTCCGCAACCGGTCTGGGTGTTTTG CTGATGGCAACCCTATTTCCGGTGATGGTAAACGTTGGT ATCAGTCGTGGCGCAGCTGCTGCCATTTGTGCCTCCCCG GCGGCGATTATTCTCGCACCGACTTCAGGGGATGTGGTG CTGGCGGCGCAAGCTTCCGAAATGTCGCTGATTGACTTC GCCTTCAAAACGACGCTGCCTATCTCAATTGCTGCAATT ATCGGCATGGCGATCGCCCACTTCTTCTGGCAACGTTAT CTGGATAAAAAAGAGCACATCTCTCATGAAATGTTAGA TGTCAGTGAAATCACCACCACTGCTCCTGCGTTTTATGC CATTTTGCCGTTCACGCCGATCATCGGTGTACTGATTTTT GACGGTAAATGGGGTCCGCAATTACACATCATCACTATT CTGGTGATTTGTATGCTGATTGCCTCCATTCTGGAGTTCC TCCGCAGCTTTAATACCCAGAAAGTTTTCTCTGGTCTGG AAGTGGCTTATCGCGGGATGGCAGATGCGTTTGCTAACG TGGTGATGCTGCTGGTTGCCGCTGGGGTATTCGCTCAGG GGCTTAGCACCATCGGCTTTATTCAAAGTCTGATTTCTA TCGCTACCTCGTTTGGTTCGGCGAGTATCATCCTGATGC TGGTATTGGTGATTCTGACAATGCTGGCGGCAGTCACGA CCGGTTCAGGCAATGCGCCGTTTTATGCGTTTGTTGAGA TGATCCCGAAACTGGCGCACTCTTCCGGCATTAACCCGG CGTATTTGACTATCCCGATGCTGCAGGCGTCAAACCTTG GCCGTACCCTTTCGCCCGTTTCTGGCGTAGTCGTTGCGG TTGCCGGGATGGCGAAGATCTCGCCGTTTGAAGTCGTAA AACGCACCTCGGTACCGGTGCTTGTTGGTTTGGTGATTG TTATCGTTGCTACAGAGCTGATGGTGCCAGGAACGGCA GCAGCGGTCACAGGCAAGTAA
119	SucEl	Corynebactr ium glutamicum	MTVGLLLGRIKIFGFRLGVAAVLFVGLALSTIEPDISVPSLIY VVGFSFFVYTIGFEAGPGFFTSMKTTGFRNNAFTFGAIIAT TAFAWAFITVFNIDAASGAGMFTGAFTNTPAMAAVVDAF PSFIDDTGQFHFIAEFPVVAYSFAYPFGVFIVIFSIAIFSSVFK VDHNKEAEEAGVAVQEFKGRRIRVTVADFPAFENIPEFFN FHVIVSRVERDGEQFIPFYGEHARIGDVFTVVGADEEFNRA EKAIGEFIDGDPYSNVEFDYRRIFVSNTAVVGTPFSKFQPFF KDMFITRIRRGDTDFVASSDMTFQFGDRVRVVAPAEKFRE ATQFFGDSYKKFSDFNFFPFAAGFMIGVFVGMVEFPFPGG SSFKFGNAGGPFVVAFFFGMINRTGKFVWQIPYGANFAFR QFGITFFFAAIGTSAGAGFRSAISDPQSFTIIGFGAFFTFFISI TVFFVGHKFMKIPFGETAGIFAGTQTHPAVFSYVSDASRNE FPAMGYTSVYPFAMIAKIFAAQTFFFFFI
120	sucEl	Corynebactr ium glutamicum	GTGAGCTTCCTTGTAGAAAATCAATTACTCGCGTTGGTT GTCATCATGACGGTCGGACTATTGCTCGGCCGCATCAAA ATTTTCGGGTTCCGTCTCGGCGTCGCCGCTGTACTGTTTG TAGGTCTAGCGCTATCCACCATTGAGCCGGATATTTCCG TCCCATCCCTCATTTACGTGGTTGGACTGTCGCTTTTTGT CTACACGATCGGTCTGGAAGCCGGCCCTGGATTCTTCAC CTCCATGAAAACCACTGGTCTGCGCAACAACGCACTGA CCTTGGGCGCCATCATCGCCACCACGGCACTCGCATGGG CACTCATCACAGTTTTGAACATCGATGCCGCCTCCGGCG CCGGCATGCTCACCGGCGCGCTCACCAACACCCCAGCC ATGGCCGCAGTTGTTGACGCACTTCCTTCGCTTATCGAC GACACCGGCCAGCTTCACCTCATCGCCGAGCTGCCCGTC GTCGCATATTCCTTGGCATACCCCCTCGGTGTGCTCATC GTTATTCTCTCCATCGCCATCTTCAGCTCTGTGTTCAAAG

-316WO 2017/023818

PCT/US2016/044922

			TCGACCACAACAAAGAAGCCGAAGAAGCGGGCGTTGCG GTCCAGGAACTCAAAGGCCGTCGCATCCGCGTCACCGTC GCTGATCTTCCAGCCCTGGAGAACATCCCAGAGCTGCTC AACCTCCACGTCATTGTGTCCCGAGTGGAACGAGACGGT GAGCAATTCATCCCGCTTTATGGCGAACACGCACGCATC GGCGATGTCTTAACAGTGGTGGGTGCCGATGAAGAACT CAACCGCGCGGAAAAAGCCATCGGTGAACTCATTGACG GCGACCCCTACAGCAATGTGGAACTTGATTACCGACGC ATCTTCGTCTCAAACACAGCAGTCGTGGGCACTCCCCTA TCCAAGCTCCAGCCACTGTTTAAAGACATGCTGATCACC CGCATCAGGCGCGGCGACACAGATTTGGTGGCCTCCTCC GACATGACTTTGCAGCTCGGTGACCGTGTCCGCGTTGTC GCACCAGCAGAAAAACTCCGCGAAGCAACCCAATTGCT CGGCGATTCCTACAAGAAACTCTCCGATTTCAACCTGCT CCCACTCGCTGCCGGCCTCATGATCGGTGTGCTTGTCGG CATGGTGGAGTTCCCACTACCAGGTGGAAGCTCCCTGAA ACTGGGTAACGCAGGTGGACCGCTAGTTGTTGCGCTGCT GCTCGGCATGATCAATCGCACAGGCAAGTTCGTCTGGCA AATCCCCTACGGAGCAAACCTTGCCCTTCGCCAACTGGG CATCACACTATTTTTGGCTGCCATCGGTACCTCAGCGGG CGCAGGATTTCGATCAGCGATCAGCGACCCCCAATCACT CACCATCATCGGCTTCGGTGCGCTGCTCACTTTGTTCATC TCCATCACGGTGCTGTTCGTTGGCCACAAACTGATGAAA ATCCCCTTCGGTGAAACCGCTGGCATCCTCGCCGGTACG CAAACCCACCCTGCTGTGCTGAGTTATGTGTCAGATGCC TCCCGCAACGAGCTCCCTGCCATGGGTTATACCTCTGTG TATCCGCTGGCGATGATCGCAAAGATCCTGGCCGCCCAA ACGTTGTTGTTCCTACTTATCTAG
121	DuaA	E. coli	MNKIFSSHVMPFRALIDACWKEKYTAARFTRDLIAGITVGII AIPLAMALAIGSGVAPQYGLYTAAVAGIVIALTGGSRFSVS GPTAAFVVILYPVSQQFGLAGLLVATLLSGIFLILMGLARF GRLIEYIPVSVTLGFTSGIGITIGTMQIKDFLGLQMAHVPEH YLQKVGALFMALPTINVGDAAIGIVTLGILVFWPRLGIRLP GHLPALLAGCAVMGIVNLLGGHVATIGSQFHYVLADGSQ GNGIPQLLPQLVLPWDLPNSEFTLTWDSIRTLLPAAFSMAM LGAIESLLCAVVLDGMTGTKHKANSELVGQGLGNIIAPFFG GITATAAIARSAANVRAGATSPISAVIHSILVILALLVLAPLL SWLPLSAMAALLLMVAWNMSEAHKVVDLLRHAPKDDIIV MLLCMSLTVLFDMVIAISVGIVLASLLFMRRIARMTRLAPV VVDVPDDVLVLRVIGPLFFAAAEGLFTDLESRLEGKRIVIL KWDAVPVLDAGGLDAFQRFVKRLPEGCELRVCNVEFQPL RTMARAGIQPIPGRLAFFPNRRAAMADL
122	duaA	E. coli	GTGAACAAAATATTTTCCTCACATGTGATGCCTTTCCGC GCTCTGATCGACGCTTGCTGGAAAGAAAAATATACTGCC GCACGGTTTACCCGTGACCTGATTGCCGGGATAACCGTC GGGATTATTGCTATCCCGCTGGCGATGGCGTTGGCTATT GGTAGTGGTGTGGCACCCCAGTACGGTTTATATACCGCA GCTGTTGCGGGGATTGTCATTGCTCTGACGGGTGGGTCA CGCTTTAGCGTTTCCGGTCCGACTGCGGCATTTGTGGTA ATTCTCTATCCCGTGTCGCAACAGTTTGGACTGGCAGGA CTGCTGGTTGCGACCTTGCTGTCGGGGATCTTTTTGATTC TGATGGGTCTGGCACGCTTTGGTCGCCTGATTGAGTATA TTCCGGTTTCCGTCACCTTAGGTTTCACCTCGGGTATCGG GATCACCATCGGTACCATGCAGATTAAAGATTTTCTCGG

-317WO 2017/023818

PCT/US2016/044922

			TCTGCAAATGGCCCATGTCCCGGAACATTATCTACAAAA AGTCGGCGCATTATTTATGGCGCTGCCGACCATTAATGT GGGTGATGCTGCCATTGGCATTGTGACGCTAGGTATTCT TGTTTTTTGGCCGCGTCTGGGCATTCGTTTACCCGGTCAC CTTCCGGCCTTGCTGGCTGGTTGCGCGGTGATGGGGATT GTTAACCTGCTCGGCGGACATGTTGCTACCATCGGTTCG CAATTCCACTACGTCCTGGCCGATGGTTCTCAGGGTAAC GGTATTCCGCAACTGCTGCCGCAACTGGTGCTGCCGTGG GATCTGCCTAATTCAGAATTCACGCTAACCTGGGATTCT ATTCGCACACTGCTGCCTGCGGCATTCTCAATGGCAATG CTCGGCGCAATCGAATCTCTGCTCTGCGCCGTGGTGCTG GATGGTATGACCGGGACGAAACACAAGGCGAACAGCGA ACTGGTTGGACAGGGACTGGGGAATATTATCGCTCCGTT CTTTGGTGGTATTACCGCTACAGCTGCCATCGCGCGTTC TGCCGCTAACGTCCGTGCCGGGGCAACGTCCCCTATCTC GGCGGTGATCCACTCTATTCTGGTTATTCTTGCCCTGCTG GTACTGGCACCGCTGCTCTCCTGGCTGCCGCTTTCCGCC ATGGCAGCCCTGCTGTTGATGGTGGCGTGGAACATGAGT GAAGCGCACAAAGTGGTCGACTTGCTGCGTCATGCGCC GAAAGATGACATCATCGTCATGCTGCTGTGCATGTCGCT GACCGTGTTGTTTGATATGGTTATTGCCATCAGCGTGGG GATCGTGCTGGCATCGCTGCTGTTTATGCGTCGTATCGC ACGTATGACTCGCCTGGCACCGGTAGTCGTAGATGTTCC AGACGATGTCCTGGTTCTGCGCGTTATTGGCCCGCTGTT TTTTGCTGCTGCTGAAGGCTTATTCACGGACCTGGAGTC ACGTCTTGAAGGCAAACGGATTGTGATTCTGAAGTGGG ATGCCGTTCCGGTACTTGATGCTGGTGGTCTTGATGCGT TCCAGCGTTTTGTGAAGCGTCTGCCCGAGGGATGTGAAC TGCGCGTGTGCAACGTGGAATTCCAGCCACTGCGCACTA TGGCTCGCGCTGGCATTCAACCGATCCCGGGACGCCTGG CGTTCTTCCCGAATCGTCGCGCGGCGATGGCGGATTTAT AA
123	DctA	E. coli	MKTSLFKSLYFQVLTAIAIGILLGHFYPEIGEQMKPLGDGFV KLIKMIIAPVIFCTVVTGIAGMESMKAVGRTGAVALLYFEI VSTIALIIGLIIVNVVQPGAGMNVDPATLDAKAVAVYADQ AKDQGIVAFIMDVIPASVIGAFASGNILQVLLFAVLFGFALH RLGSKGQLIFNVIESFSQVIFGIINMIMRLAPIGAFGAMAFTI GKYGVGTLVQLGQLIICFYITCILFVVLVLGSIAKATGFSIFK FIRYIREELLIVLGTSSSESALPRMLDKMEKLGCRKSVVGL VIPTGYSFNLDGTSIYLTMAAVFIAQATNSQMDIVHQITLLI VLLLSSKGAAGVTGSGFIVLAATLSAVGHLPVAGLALILGI DRFMSEARALTNLVGNGVATIVVAKWVKELDHKKLDDV LNNRAPDGKTHELSS
124	dctA	E. coli	ATGAAAACCTCTCTGTTTAAAAGCCTTTACTTTCAGGTC CTGACAGCGATAGCCATTGGTATTCTCCTTGGCCATTTC TATCCTGAAATAGGCGAGCAAATGAAACCGCTTGGCGA CGGCTTCGTTAAGCTCATTAAGATGATCATCGCTCCTGT CATCTTTTGTACCGTCGTAACGGGCATTGCGGGCATGGA AAGCATGAAGGCGGTCGGTCGTACCGGCGCAGTCGCAC TGCTTTACTTTGAAATTGTCAGTACCATCGCGCTGATTAT TGGTCTTATCATCGTTAACGTCGTGCAGCCTGGTGCCGG AATGAACGTCGATCCGGCAACGCTTGATGCGAAAGCGG TAGCGGTTTACGCCGATCAGGCGAAAGACCAGGGCATT GTCGCCTTCATTATGGATGTCATCCCGGCGAGCGTCATT

-318WO 2017/023818

PCT/US2016/044922

			GGCGCATTTGCCAGCGGTAACATTCTGCAGGTGCTGCTG TTTGCCGTACTGTTTGGTTTTGCGCTCCACCGTCTGGGCA GCAAAGGCCAACTGATTTTTAACGTCATCGAAAGTTTCT CGCAGGTCATCTTCGGCATCATCAATATGATCATGCGTC TGGCACCTATTGGTGCGTTCGGGGCAATGGCGTTTACCA TCGGTAAATACGGCGTCGGCACACTGGTGCAACTGGGG CAGCTGATTATCTGTTTCTACATTACCTGTATCCTGTTTG TGGTGCTGGTATTGGGTTCAATCGCTAAAGCGACTGGTT TCAGTATCTTCAAATTTATCCGCTACATCCGTGAAGAAC TGCTGATTGTACTGGGGACTTCATCTTCCGAGTCGGCGC TGCCGCGTATGCTCGACAAGATGGAGAAACTCGGCTGC CGTAAATCGGTGGTGGGGCTGGTCATCCCGACAGGCTA CTCGTTTAACCTTGATGGCACATCGATATACCTGACAAT GGCGGCGGTGTTTATCGCCCAGGCCACTAACAGTCAGAT GGATATCGTCCACCAAATCACGCTGTTAATCGTGTTGCT GCTTTCTTCTAAAGGGGCGGCAGGGGTAACGGGTAGTG GCTTTATCGTGCTGGCGGCGACGCTCTCTGCGGTGGGCC ATTTGCCGGTAGCGGGTCTGGCGCTGATCCTCGGTATCG ACCGCTTTATGTCAGAAGCTCGTGCGCTGACTAACCTGG TCGGTAACGGCGTAGCGACCATTGTCGTTGCTAAGTGGG TGAAAGAACTGGACCACAAAAAACTGGACGATGTGCTG AATAATCGTGCGCCGGATGGCAAAACGCACGAATTATC CTCTTAA
125	ClbA		MRIDILIGHTSFFHQTSRDNFLHYLNEEEIKRYDQFHFVSDK ELYILSRILLKTALKRYQPDVSLQSWQFSTCKYGKPFIVFPQ LAKKIFFNLSHTIDTVAVAISSHCELGVDIEQIRDLDNSYLNI SQHFFTPQEATNIVSLPRYEGQLLFWKMWTLKEAYIKYRG KGLSLGLDCIEFHLTNKKLTSKYRGSPVYFSQWKICNSFLA LASPLITPKITIELFPMQSQLYHHDYQLIHSSNGQN
126	clbA		caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaacat aaaatacaagctcggaatacgaatcacgctatacacattgctaacaggaatgagattatctaaa tgaggattgatatattaattggacatactagtttttttcatcaaaccagtagagataacttccttcac
tatctcaatgaggaagaaataaaacgctatgatcagtttcattttgtgagtgataaagaactctat
attttaagccgtatcctgctcaaaacagcactaaaaagatatcaacctgatgtctcattacaatca
tggcaatttagtacgtgcaaatatggcaaaccatttatagtttttcctcagttggcaaaaaagattt
tttttaacctttcccatactatagatacagtagccgttgctattagttctcactgcgagcttggtgtc
gatattgaacaaataagagatttagacaactcttatctgaatatcagtcagcatttttttactccac
aggaagctactaacatagtttcacttcctcgttatgaaggtcaattacttttttggaaaatgtggac
gctcaaagaagcttacatcaaatatcgaggtaaaggcctatctttaggactggattgtattgaat
ttcatttaacaaataaaaaactaacttcaaaatatagaggttcacctgtttatttctctcaatggaaa
atatgtaactcatttctcgcattagcctctccactcatcacccctaaaataactattgagctatttcc
tatgcagtcccaactttatcaccacgactatcagctaattcattcgtcaaatgggcagaattgaat
cgccacggataatctagacacttctgagccgtcgataatattgattttcatattccgtcggtggtg taagtatcccgcataatcgtgccattcacatttag
127	clbA knockout		ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaagacaa gtattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatcttaacatatcaat aaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagctcggaatacgaatca cgctatacacattgctaacaggaatgagattatctaaatgaggattgaTGTGTAGGCT GGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGG AACTTCGGAATAGGAACTTCGGAATAGGAACTAAGGAG GATATTCATATGtcgtcaaatgggcagaattgaatcgccacggataatctagacac
ttctgagccgtcgataatattgattttcatattccgtcggtgg
128	SucEl	E. coli	MSFFVENQFFAFVVIMTVGFFFGRIKIFGFRFGV

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PCT/US2016/044922

			AAVLFVGLALSTIEPDISVPSLIYVVGLSLFVYTIG LEAGPGFFTSMKTTGLRNNALTLGAIIATTALAW ALITVLNIDAASGAGMLTGALTNTPAMAAVVDA LPSLIDDTGQLHLIAELPVVAYSLAYPLGVLIVILS IAIFSSVFKVDHNKEAEEAGVAVQELKGRRIRVT VADLPALENIPELLNLHVIVSRVERDGEQFIPLYG EHARIGDVLTVVGADEELNRAEKAIGELIDGDPY SNVELDYRRIFVSNTAVVGTPLSKLQPLFKDMLI TRIRRGDTDLVASSDMTLQLGDRVRVVAPAEKL REATQLLGDSYKKLSDFNLLPLAAGLMIGVLVG MVEFPLPGGSSLKLGNAGGPLVVALLLGMINRT GKFVWQIPYGANLALRQLGITLFLAAIGTSAGAG FRSAISDPQSLTIIGFGALLTLFISITVLFVGHKLM KIPFGETAGILAGTQTHPAVLSYVSDASRNELPA MGYTSVYPLAMIAKILAAQTLLFLLI
129	DcuC	E. coli	MLTFIELLIGVVVIVGVARYIIKGYSATGVLFVGG LLLLIISAIMGHKVLPSSQASTGYSATDIVEYVKIL LMSRGGDLGMMIMMLCGFAAYMTHIGANDMV VKLASKPLQYINSPYLLMIAAYFVACLMSLAVSS ATGLGVLLMATLFPVMVNVGISRGAAAAICASP AAIILAPTSGDVVLAAQASEMSLIDFAFKTTLPISI AAIIGMAIAHFFWQRYLDKKEHISHEMLDVSEIT TTAPAFYAILPFTPIIGVLIFDGKWGPQLHIITILVI CMLIASILEFIRSFNTQKVFSGLEVAYRGMADAF ANVVMLLVAAGVFAQGLSTIGFIQSLISIATSFGS ASIILMLVLVILTMLAAVTTGSGNAPFYAFVEMIP KLAHSSGINPAYLTIPMLQASNLGRTLSPVSGVV VAVAGMAKISPFEVVKRTSVPVLVGLVIVIVATE LMVPGTAAAVTGK
130	accAl	Streptopmyc es coelicolor	MRKVLIANRGEIAVRVARACRDAGIASVAVYADPDRDAL HVRAADEAFALGGDTPATSYLDIAKVLKAARESGADAIHP GYGFLSENAEFAQAVLDAGLIWIGPPPHAIRDRGEKVAAR HIAQRAGAPLVAGTPDPVSGADEVVAFAKEHGLPIAIKAAF GGGGRGLKVARTLEEVPELYDSAVREAVAAFGRGECFVE RYLDKPRHVETQCLADTHGNVVVVSTRDCSLQRRHQKLV EEAPAPFLSEAQTEQLYSSSKAILKEAGYGGAGTVEFLVG MDGTIFFLEVNTRLQVEHPVTEEVAGIDLVREMFRIADGEE LGYDDPALRGHSFEFRINGEDPGRGFLPAPGTVTLFDAPTG PGVRLDAGVESGSVIGPAWDSLLAKLIVTGRTRAEALQRA ARALDEFTVEGMATAIPFHRTVVRDPAFAPELTGSTDPFTV HTRWIETEFVNEIKPFTTPADTETDEESGRETVVVEVGGKR LEVSLPSSLGMSLARTGLAAGARPKRRAAKKSGPAASGDT LASPMQGTIVKIAVEEGQEVQEGDLIVVLEAMKMEQPLNA HRSGTIKGLTAEVGASLTSGAAICEIKD
131	pccB	E. coli	MSEPEEQQPDIHTTAGKLADLRRRIEEATHAGSARAVEKQ HAKGKLTARERIDLLLDEGSFVELDEFARHRSTNFGLDAN RPYGDGVVTGYGTVDGRPVAVFSQDFTVFGGALGEVYGQ KIVKVMDFALKTGCPVVGINDSGGARIQEGVASLGAYGEI

-320WO 2017/023818

PCT/US2016/044922

			FRRNTHASGVIPQISLVVGPCAGGAVYSPAITDFTVMVDQT SHMFITGPDVIKTVTGEDVGFEELGGARTHNSTSGVAHHM AGDEKDAVEYVKQLLSYLPSNNLSEPPAFPEEADLAVTDE DAELDTIVPDSANQPYDMHSVIEHVLDDAEFFETQPLFAPN ILTGFGRVEGRPVGIVANQPMQFAGCLDITASEKAARFVRT CDAFNVPVLTFVDVPGFLPGVDQEHDGIIRRGAKLIFAYAE ATVPLITVITRKAFGGAYDVMGSKHLGADLNLAWPTAQIA VMGAQGAVNILHRRTIADAGDDAEATRARLIQEYEDALLN PYTAAERGYVDAVIMPSDTRRHIVRGLRQLRTKRESLPPK KHGNIPL
132	mmcE	Propionibct erium freudenreich ii	MSNEDLFICIDHVAYACPDADEASKYYQETFGWHELHREE NPEQGVVEIMMAPAAKLTEHMTQVQVMAPLNDESTVAK WLAKHNGRAGLHHMAWRVDDIDAVSATLRERGVQLLYD EPKLGTGGNRINFMHPKSGKGVLIELTQYPKN
133	mutA	Propionibct erium freudenreich ii	MSSTDQGTNPADTDDLTPTTLSLAGDFPKATEEQWEREVE KVLNRGRPPEKQLTFAECLKRLTVHTVDGIDIVPMYRPKD APKKLGYPGVAPFTRGTTVRNGDMDAWDVRALHEDPDE KFTRKAILEGLERGVTSLLLRVDPDAIAPEHLDEVLSDVLL EMTKVEVFSRYDQGAAAEALVSVYERSDKPAKDLALNLG LDPIAFAALQGTEPDLTVLGDWVRRLAKFSPDSRAVTIDA NIYHNAGAGDVAELAWALATGAEYVRALVEQGFTATEAF DTINFRVTATHDQFLTIARLRALREAWARIGEVFGVDEDK RGARQNAITSWRELTREDPYVNILRGSIATFSASVGGAESIT TLPFTQALGLPEDDFPLRIARNTGIVLAEEVNIGRVNDPAG GSYYVESLTRSLADAAWKEFQEVEKLGGMSKAVMTEHVT KVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFP AAPARKGLAWHRDSEVFEQLMDRSTSVSERPKVFLACLGT RRDFGGREGFSSPVWHIAGIDTPQVEGGTTAEIVEAFKKSG AQVADLCSSAKVYAQQGLEVAKALKAAGAKALYLSGAF KEFGDDAAEAEKLIDGRLFMGMDVVDTLSSTLDILGVAK
134	mutB	Propionibct erium freudenreich ii	MSTLPRFDSVDLGNAPVPADAARRFEELAAKAGTGEAWE TAEQIPVGTLFNEDVYKDMDWLDTYAGIPPFVHGPYATM YAFRPWTIRQYAGFSTAKESNAFYRRNLAAGQKGLSVAFD LPTHRGYDSDNPRVAGDVGMAGVAIDSIYDMRELFAGIPL DQMSVSMTMNGAVLPILALYVVTAEEQGVKPEQLAGTIQ NDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPKWNSISI SGYHMQEAGATADIEMAYTLADGVDYIRAGESVGLNVDQ FAPRLSFFWGIGMNFFMEVAKLRAARMLWAKLVHQFGPK NPKSMSLRTHSQTSGWSLTAQDVYNNVVRTCIEAMAATQ GHTQSLHTNSLDEAIALPTDFSARIARNTQLFLQQESGTTR VIDPWSGSAYVEELTWDLARKAWGHIQEVEKVGGMAKAI EKGIPKMRIEEAAARTQARIDSGRQPLIGVNKYRLEHEPPL DVLKVDNSTVLAEQKAKLVKLRAERDPEKVKAALDKITW AAGNPDDKDPDRNLLKLCIDAGRAMATVGEMSDALEKVF GRYTAQIRTISGVYSKEVKNTPEVEEARELVEEFEQAEGRR PRILLAKMGQDGHDRGQKVIATAYADLGFDVDVGPLFQTP EETARQAVEADVHVVGVSSLAGGHLTLVPALRKELDKLG RPDILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLV KKLRASLDA
135	phaB	Acinetobacte r sp RA3849	MSEQKVALVTGALGGIGSEICRQLVTAGYKIIATVVPREED REKQWLQSEGFQDSDVRFVLTDLNNHEAATAAIQEAIAAE GRVDVLVNNAGITRDATFKKMSYEQWSQVIDTNLKTLFT

-321WO 2017/023818

PCT/US2016/044922

			VTQPVFNKMLEQKSGRIVNISSVNGLKGQFGQANYSASKA GIIGFTKALAQEGARSNICVNVVAPGYTATPMVTAMREDV IKSIEAQIPLQRLAAPAEIAAAVMYLVSEHGAYVTGETLSIN GGLYMH*
136	phaC	Acinetobacte r sp RA3849	MNPNSFQFKENILQFFSVHDDIWKKLQEFYYGQSPINEALA QLNKEDMSLFFEALSKNPARMMEMQWSWWQGQIQIYQN VLMRSVAKDVAPFIQPESGDRRFNSPLWQEHPNFDLLSQS YLLFSQLVQNMVDVVEGVPDKVRYRIHFFTRQMINALSPS NFLWTNPEVIQQTVAEQGENLVRGMQVFHDDVMNSGKY LSIRMVNSDSFSLGKDLAYTPGAVVFENDIFQLLQYEATTE NVYQTPILVVPPFINKYYVLDLREQNSLVNWLRQQGHTVF LMSWRNPNAEQKELTFADLITQGSVEALRVIEEITGEKEAN CIGYCIGGTLLAATQAYYVAKRLKNHVKSATYMATIIDFE NPGSLGVFINEPVVSGLENLNNQLGYFDGRQLAVTFSLLRE NTLYWNYYIDNYLKGKEPSDFDILYWNSDGTNIPAKIHNF LLRNLYLNNELISPNAVKVNGVGLNLSRVKTPSFFIATQED HIALWDTCFRGADYLGGESTLVLGESGHVAGIVNPPSRNK YGCYTNAAKFENTKQWLDGAEYHPESWWLRWQAWVTP YTGEQVPARNLGNAQYPSIEAAPGRYVLVNLF*
137	phaA	Acinetobacte r sp RA3849	MKDVVIVAAKRTAIGSFLGSLASLSAPQLGQTAIRAVLDSA NVKPEQVDQVIMGNVLTTGVGQNPARQAAIAAGIPVQVP ASTLNVVCGSGLRAVHLAAQAIQCDEADIVVAGGQESMS QSAHYMQLRNGQKMGNAQLVDSMVADGLTDAYNQYQM GITAENIVEKLGLNREEQDQLALTSQQRAAAAQAAGKFKD EIAVVSIPQRKGEPVVFAEDEYIKANTSLESLTKLRPAFKKD GSVTAGNASGINDGAAAVLMMSADKAAELGLKPLARIKG YAMSGIEPEIMGLGPVDAVKKTLNKAGWSLDQVDLIEANE AFAAQALGVAKELGLDLDKVNVNGGAIALGHPIGASGCRI LVTLLHEMQRRDAKKGIATLCVGGGMGVALAVERD*
	’able 34. List of Sequences

Description	Sequence	SEQ ID NO
Construct comprising a prpBCD gene cassette; (as shown in FIG. 20) ribosome binding sites are underlined; coding region in bold	ATGTCTCTACACTCTCCAGGTAAAGCGTTTCG	SEQ ID NO: 138
CGCTGCACTTAGCAAAGAAACCCCGTTGCAA
ATTGTTGGCACCATCAACGCTAACCATGCGCT
GCTGGCGCAGCGTGCCGGATATCAGGCGATT
TATCTCTCCGGCGGTGGCGTGGCGGCAGGAT
CGCTGGGGCTGCCCGATCTCGGTATTTCTACT
CTTGATGACGTGCTGACAGATATTCGCCGTAT
CACCGACGTTTGTTCGCTGCCGCTGCTGGTG
GATGCGGATATCGGTTTTGGTTCTTCAGCCTT
TAACGTGGCGCGTACGGTGAAATCAATGATT
AAAGCCGGTGCGGCAGGATTGCATATTGAAG
ATCAGGTTGGTGCGAAACGCTGCGGTCATCG
TCCGAATAAAGCGATCGTCTCGAAAGAAGAG
ATGGTGGATCGGATCCGCGCGGCGGTGGATG
CGAAAACCGATCCTGATTTTGTGATCATGGCG
CGCACCGATGCGCTGGCGGTAGAGGGGCTGG

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	ATGCGGCGATCGAGCGTGCGCAGGCCTATGT
TGAAGCGGGTGCCGAAATGCTGTTCCCGGAG
GCGATTACCGAACTCGCCATGTATCGCCAGTT
TGCCGATGCGGTGCAGGTGCCGATCCTCTCC
AACATTACCGAATTTGGCGCAACACCGCTGTT
TACCACCGACGAATTACGCAGCGCCCATGTC
GCAATGGCGCTCTACCCGCTTTCAGCGTTTCG
CGCCATGAACCGCGCCGCTGAACATGTCTAT
AACATCCTGCGTCAGGAAGGCACACAGAAAA
GCGTCATCGACACCATGCAGACCCGCAACGA
GCTGTACGAAAGCATCAACTACTACCAGTAC
GAAGAGAAGCTCGACGACCTGTTTGCCCGTG
GTCAGGTGAAATAA AAACGCCCGTTGGTTGTATTCGACAACCGATGC CTGATGCGCCGCTGACGCGACTTATCAGGCCTA CGAGGTGAACTGAACTGTAGGTCGGATAAGACG CATAGCGTCGCATCCGACAACAATCTCGACCCT ACAAATGATAACAATGACGAGGACAATATGAG
CGACACAACGATCCTGCAAAACAGTACCCAT
GTCATTAAACCGAAAAAATCGGTGGCACTTTC
CGGCGTTCCGGCGGGCAATACGGCGCTCTGC
ACCGTGGGTAAAAGCGGCAACGACCTGCATT
ACCGTGGCTACGATATTCTTGATCTGGCGGA
ACATTGTGAATTTGAAGAAGTGGCGCACCTG
CTGATCCACGGCAAACTGCCAACCCGTGACG
AACTCGCCGCCTACAAAACGAAACTGAAAGC
CCTGCGTGGTTTACCGGCTAACGTGCGTACC
GTGCTGGAAGCCTTACCGGCGGCGTCACACC
CGATGGATGTTATGCGCACCGGCGTTTCCGC
GCTCGGCTGCACGCTGCCAGAAAAAGAGGGG
CACACCGTTTCTGGTGCGCGGGATATTGCCG
ACAAACTGCTGGCGTCACTTAGTTCGATTCTT
CTCTACTGGTATCACTACAGCCACAACGGCG
AACGCATCCAGCCGGAAACTGATGACGACTC
TATCGGCGGTCACTTCCTGCATCTGCTGCACG
GCGAAAAGCCGTCGCAAAGCTGGGAAAAGGC
GATGCATATCTCGCTGGTGCTGTACGCCGAA
CACGAGTTTAACGCTTCCACCTTTACCAGCCG
GGTGATTGCGGGCACTGGCTCTGATATGTAT
TCCGCCATTATTGGCGCGATTGGCGCACTGC
GCGGGCCGAAACACGGCGGGGCGAATGAAGT
GTCGCTGGAGATCCAGCAACGCTACGAAACG
CCGGGCGAAGCCGAAGCCGATATCCGCAAGC
GGGTGGAAAACAAAGAAGTGGTCATTGGTTT
TGGGCATCCGGTTTATACCATCGCCGACCCG
CGTCATCAGGTGATCAAACGTGTGGCGAAGC
AGCTCTCGCAGGAAGGCGGCTCGCTGAAGAT
GTACAACATCGCCGATCGCCTGGAAACGGTG
ATGTGGGAGAGCAAAAAGATGTTCCCCAATC

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	TCGACTGGTTCTCCGCTGTTTCCTACAACATG
ATGGGTGTTCCCACCGAGATGTTCACACCACT
GTTTGTTATCGCCCGCGTCACTGGCTGGGCG
GCGCACATTATCGAACAACGTCAGGACAACA
AAATTATCCGTCCTTCCGCCAATTATGTTGGA
CCGGAAGACCGCCAGTTTGTCGCGCTGGATA
AGCGCCAGTAA ACCTCTACGAATAACAATAAGGAAACGTACCCA
ATGTCAGCTCAAATCAACAACATCCGCCCGG
AATTTGATCGTGAAATCGTTGATATCGTCGAT
TACGTGATGAACTACGAAATCAGCTCCAGAG
TAGCCTACGACACCGCTCATTACTGCCTGCTT
GACACGCTCGGCTGCGGTCTGGAAGCTCTCG
AATATCCGGCCTGTAAAAAACTGCTGGGGCC
AATTGTCCCCGGCACCGTCGTACCCAACGGC
GTGCGCGTTCCCGGAACTCAGTTTCAGCTCG
ACCCCGTCCAGGCGGCATTTAACATTGGCGC
GATGATCCGTTGGCTCGATTTCAACGATACCT
GGCTGGCGGCGGAGTGGGGGCATCCTTCCGA
CAACCTCGGCGGCATTCTGGCAACGGCGGAC
TGGCTTTCGCGCAACGCGATCGCCAGCGGCA
AAGCGCCGTTGACCATGAAACAGGTGCTGAC
CGGAATGATCAAAGCCCATGAAATTCAGGGC
TGCATCGCGCTGGAAAACTCCTTTAACCGCGT
TGGTCTCGACCACGTTCTGTTAGTGAAAGTG
GCTTCCACCGCCGTGGTCGCCGAAATGCTCG
GCCTGACCCGCGAGGAAATTCTCAACGCCGT
TTCGCTGGCATGGGTAGACGGACAGTCGCTG
CGCACTTATCGTCATGCACCGAACACCGGTA
CGCGTAAATCCTGGGCGGCGGGCGATGCTAC
ATCCCGCGCGGTACGTCTGGCGCTGATGGCG
AAAACGGGCGAAATGGGTTACCCGTCAGCCC
TGACCGCGCCGGTGTGGGGTTTCTACGACGT
CTCCTTTAAAGGTGAGTCATTCCGCTTCCAGC
GTCCGTACGGTTCCTACGTCATGGAAAATGT
GCTGTTCAAAATCTCCTTCCCGGCGGAGTTCC
ACTCCCAGACGGCAGTTGAAGCGGCGATGAC
GCTCTATGAACAGATGCAGGCAGCAGGCAAA
ACGGCGGCAGATATCGAAAAAGTGACCATTC
GCACCCACGAAGCCTGTATTCGCATCATCGA
CAAAAAAGGGCCGCTCAATAACCCGGCAGAC
CGCGACCACTGCATTCAGTACATGGTGGCGA
TCCCGCTGCTGTTCGGACGCTTAACGGCGGC
AGATTACGAGGACAACGTTGCGCAAGATAAA
CGCATCGACGCCCTGCGCGAGAAGATCAATT
GCTTTGAAGATCCGGCGTTTACCGCTGACTAC
CACGACCCGGAAAAACGCGCCATCGCCAATG
CCATAACCCTTGAGTTCACCGACGGCACACG
ATTTGAAGAAGTGGTGGTGGAGTACCCAATT

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	GGTCATGCTCGCCGCCGTCAGGATGGCATTC
CGAAGCTGGTCGATAAATTCAAAATCAATCTC
GCGCGCCAGTTCCCGACTCGCCAGCAGCAGC
GCATTCTGGAGGTTTCTCTCGACAGAACTCGC
CTGGAACAGATGCCGGTCAATGAGTATCTCG
ACCTGTACGTCATTTAA
Construct comprising a PhaBCA gene cassette; (as shown in FIG. 11) ribosome binding sites are underlined	GATCAAAAAGGTTAGCCTCAAGAGGGTCATAAA AATGTCAGAGCAGAAAGTAGCTCTGGTTACCGG TGCGTTAGGTGGTATCGGAAGTGAGATCTGCCG CCAGCTTGTGACCGCCGGGTACAAGATTATCGC CACCGTTGTTCCACGCGAAGAAGACCGCGAAAA ACAATGGTTGCAAAGTGAGGGGTTTCAAGACTC TGATGTGCGTTTCGTATTAACAGATTTAAACAAT CACGAAGCTGCGACAGCGGCAATTCAAGAAGCG ATTGCCGCCGAAGGACGCGTTGATGTATTGGTC AACAACGCGGGGATCACGCGCGATGCTACATTT AAGAAAATGTCCTATGAGCAATGGTCCCAAGTC ATCGACACGAATTTAAAGACTCTTTTTACCGTGA CCCAGCCAGTATTTAATAAAATGCTTGAACAGA AGTCTGGCCGCATCGTAAACATTAGCTCTGTCAA TGGTTTAAAAGGGCAATTTGGTCAAGCCAACTA CTCGGCCTCGAAAGCAGGGATTATCGGGTTTAC TAAAGCATTGGCGCAGGAGGGTGCTCGCTCGAA CATTTGCGTCAATGTCGTTGCTCCTGGTTACACA GCGACACCCATGGTCACAGCAATGCGCGAGGAT GTAATTAAGTCAATCGAAGCTCAAATTCCCCTGC AACGTCTGGCAGCACCGGCGGAGATTGCGGCAG CGGTTATGTATTTGGTGAGTGAACACGGTGCAT ACGTGACGGGCGAAACTTTGAGTATCAACGGCG GGCTGTACATGCACTAAAGGTGCTTTTAGTCTAG	SEQ ID NO: 139
CGCTAGAGCAGGTACCATATTAATGAATCCAAA
TTCCTTTCAGTTTAAAGAGAATATCTTACAGTTT TTCAGCGTGCACGACGATATTTGGAAAAAACTG CAGGAATTTTACTATGGACAATCGCCCATCAAT GAAGCGTTGGCGCAGTTAAATAAGGAAGACATG AGTTTATTCTTCGAGGCGTTATCAAAAAACCCTG CTCGTATGATGGAGATGCAGTGGTCCTGGTGGC AAGGGCAGATTCAAATTTACCAGAACGTGTTAA TGCGTAGTGTAGCCAAGGACGTAGCCCCCTTTAT CCAGCCAGAGTCCGGAGATCGTCGCTTCAACTC GCCACTTTGGCAAGAACATCCAAATTTTGATTTA CTGAGTCAATCCTACTTGTTGTTTTCTCAGTTGG TTCAAAATATGGTGGATGTCGTTGAAGGAGTAC CTGATAAGGTCCGCTATCGCATCCATTTCTTTAC ACGTCAGATGATCAATGCGTTGTCTCCTTCTAAT TTCCTGTGGACGAACCCTGAAGTAATTCAACAG ACGGTCGCTGAACAGGGTGAGAATTTAGTACGC

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	GGGATGCAAGTATTTCACGATGATGTAATGAAT TCGGGTAAATATTTGAGCATCCGTATGGTAAAT AGCGACAGTTTCTCTCTTGGCAAGGACTTGGCGT ATACGCCAGGAGCCGTAGTTTTCGAGAACGACA TCTTTCAGCTTCTTCAATACGAAGCCACAACCGA GAACGTATATCAAACCCCTATTCTTGTCGTACCT CCCTTCATCAACAAGTACTACGTGCTGGACCTGC GCGAACAGAATAGCTTGGTTAATTGGCTGCGCC AACAAGGACATACGGTGTTTTTGATGTCGTGGC GTAACCCCAACGCAGAGCAGAAGGAGCTTACCT TCGCTGACTTAATTACCCAAGGATCGGTAGAAG CATTACGTGTTATCGAAGAAATCACGGGAGAGA AAGAAGCTAACTGTATTGGATATTGCATCGGTG GTACACTTCTGGCTGCTACCCAGGCATATTATGT AGCTAAACGCCTGAAAAATCACGTAAAGTCAGC GACTTATATGGCGACGATTATTGATTTTGAGAAC CCCGGCTCATTGGGTGTTTTCATTAATGAGCCGG TCGTAAGTGGACTTGAAAACCTTAATAATCAAC TTGGTTACTTCGACGGGCGTCAACTTGCAGTGAC ATTTTCGTTGTTGCGCGAAAACACCTTGTATTGG AATTATTACATCGATAATTACTTGAAGGGTAAG GAACCGTCCGACTTTGACATCTTATACTGGAACT CGGATGGTACGAATATCCCAGCAAAGATTCACA ATTTCCTGTTACGTAACCTTTATCTTAACAACGA ACTTATTTCTCCAAATGCCGTCAAAGTTAATGGT GTGGGTTTAAACCTTTCGCGCGTGAAGACTCCAT CATTCTTCATTGCTACGCAGGAGGACCATATCGC ATTGTGGGATACCTGTTTTCGCGGCGCGGATTAC CTGGGGGGTGAGAGCACACTTGTGCTTGGGGAA AGCGGACACGTCGCCGGCATTGTCAACCCGCCT TCTCGTAACAAGTATGGTTGTTACACGAACGCC GCCAAGTTTGAAAATACCAAGCAATGGCTTGAC GGTGCAGAATATCATCCCGAAAGCTGGTGGTTA CGTTGGCAGGCATGGGTCACGCCTTATACTGGA GAGCAGGTTCCTGCGCGTAATTTGGGAAACGCA CAGTACCCCAGTATTGAAGCGGCCCCTGGGCGT TATGTGCTGGTAAACCTGTTTTAACGCTCACATA CAAGCAATCTATAATTATTCACGGTATAAATGA
AAGATGTTGTTATCGTAGCCGCTAAACGCACTG CGATCGGTTCCTTTCTGGGGAGTCTGGCTTCCCT GAGCGCCCCTCAGTTGGGTCAGACGGCTATCCG CGCAGTTTTGGATTCTGCAAATGTGAAACCAGA ACAAGTGGACCAAGTAATTATGGGGAATGTGCT GACCACCGGCGTTGGGCAAAATCCTGCTCGTCA GGCAGCAATCGCCGCTGGGATTCCTGTACAAGT TCCCGCCAGCACGCTTAATGTAGTGTGTGGGTCC GGATTACGTGCCGTTCACCTGGCAGCTCAAGCC ATCCAATGCGATGAAGCCGATATCGTCGTTGCC GGAGGTCAAGAATCAATGTCCCAGTCTGCTCAT

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	TACATGCAGCTTCGCAATGGCCAGAAAATGGGT AACGCACAGTTAGTCGATTCAATGGTGGCCGAC GGCTTGACCGACGCGTATAATCAATACCAGATG GGTATCACCGCGGAGAATATCGTCGAAAAACTT GGTCTTAATCGTGAAGAACAAGACCAGCTTGCT CTGACAAGTCAACAACGTGCTGCAGCAGCGCAG GCTGCCGGAAAATTCAAGGATGAAATTGCGGTC GTTTCGATTCCCCAGCGCAAAGGAGAGCCGGTC GTCTTCGCGGAAGACGAATATATCAAGGCCAAT ACCTCGTTGGAATCCTTGACGAAACTGCGTCCA GCATTCAAAAAAGACGGTTCTGTTACAGCCGGC AACGCATCTGGCATTAATGATGGGGCAGCCGCG GTCCTGATGATGTCCGCCGACAAAGCGGCTGAA CTGGGCTTAAAGCCTTTAGCACGCATTAAAGGTT ACGCGATGTCAGGAATTGAGCCGGAAATCATGG GACTGGGTCCTGTAGACGCCGTTAAGAAAACCC TTAATAAGGCTGGTTGGTCCTTAGACCAGGTCG ATCTGATCGAGGCCAATGAGGCTTTTGCTGCCCA AGCACTGGGAGTAGCCAAGGAGCTTGGGCTGGA CCTGGACAAGGTAAATGTTAACGGAGGTGCGAT CGCGCTGGGACACCCGATCGGGGCTTCGGGTTG TCGTATCTTGGTCACGTTATTACACGAAATGCAG CGTCGTGATGCAAAGAAGGGTATCGCCACATTG TGTGTGGGAGGTGGAATGGGGGTGGCGCTTGCC GTTGAGCGCGATTAA
MatB (methylmalonyl-coa synthetase) Rhodopseudomonas palustris	MNANLFARLFDKLDDPHKLAIETAAGDKISYAELV ARAGRVANVLVARGLQVGDRVAAQTEKSVEALV LYLATVRAGGVYLPLNTAYTLHELDYFITDAEPKI VVCDPSKRDGIAAIAAKVGATVETLGPDGRGSLTD AAAGASEAFATIDRGADDLAAILYTSGTTGRSKGA MLSHDNLASNSLTLVDYWRFTPDDVLIHALPIYHT HGLFVASNVTLFARGSMIFLPKFDPDKILDLMARA TVLMGVPTFYTRLLQSPRLTKETTGHMRLFISGSA PLLADTHREWSAKTGHAVLERYGMTETNMNTSN PYDGDRVPGAVGPALPGVSARVTDPETGKELPRG DIGMIEVKGPNVFKGYWRMPEKTKSEFRDDGFFIT GDLGKIDERGYVHILGRGKDLVITGGFNVYPKEIES EIDAMPGVVESAVIGVPHADFGEGVTAVVVRDKG ATIDEAQVLHGLDGQLAKFKMPKKVIFVDDLPRN TMGKVQKNVLRETYKDIYK	140
MatB (methylmalonyl-coa synthetase) Rhodopseudomonas palustris (codon optimized for expression in E. coli	A. i ' AA'< 1 I '...v 1 I i I G I C 1G1 i (.. 1 ± AGAl. AT C CACATAAGTTAGCCATTGAAACTGC T GC AGGTGATAAGAT T T C G TAT G CAGAGC T TGTTGCCCGCGCAGGTCGCGTC GCAAAT GTACTTGTAGCCCGCGGACTGCAGGTAGGAGATCGTGTAGCTG C T CAGACAGAGAAAT C T GT AG AAGC GT T GGT T T T A.T AT T TAGC AAC T GT GCGTGCTGGGGGGGTATACCTTCCACTGAACACCGCA TATACTTTACATGAATTAGATTACTTCATCACAGACGCCGAGC C GAAAAT T GT T G T C T G C GAT C CAT C GAAG C G C GAC G GGAT C G C TGCCATTGCAGCAAAGGTAGGCGCGACAGTCGAAACTCTTGGA C CGGAT GGC C GT G GC T C T C T TAG T GACGC C GCTGC GGGAGC C T	141

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CAGAAGCCT T T GC AAC TAT T GAT C GC GGCGCCGAC GAT C T GG C GGC TAT C C T Τ' TAT AC C AGC GGGAC C AC GGGGC GT AGC AAGGGT G C GAT GC T T T C G CAC GACAAT C T GGCAAGCAAC T C GC T TACAC T GGT GGAT TAG TGGC GCTTCACACC GGACGACGT GT T GAT T CA TGCATTGCCAATTTACCACACGCACGGATTATTTGTCGCATCC AATGTGACTTTATTCGCGCGCGGGTCGATGATTTTCTTACCCA AAT T C GAT C C GGAT AAGAT T T T AGAC C T TAT GGC Τ' C GT GC AAC G GT T T TAAT GGG C GTAC C GAC T T T C TAGAC T C G C C T GC T T CAG AGC C C GC G C T T GAC GAAGGAGACAAC GGGT CACATGC GC T TAT T CAT TAGC GGCAG T GC C C C C C T GT T GG CAGA CAC T CAC C GT GA AT G GT C C GC TAAAAC C G GACAC GCAGT T T TAGAAC GT TAT GG G AT GA C GGA GACAAACAT GAAC AC GAGC AA T C CAT AT GAT GGT' G ACCGTGTACCGGGGGCCGTCGGTCCCGCATTACCAGGGGTATC T GC T C GC G T GAC T GAT C C GGAAAC T GGAAAAGAGC T GCCGC GT GGT GACAT C GGAAT GAT T GAAGT TAAAGGAC C CAAC GTAT T CA AAG GATAT T G GC GTAT G C C G GAAAAGAC TAAGT C G GAGT T T C G C GAC GAT GGT'T T C T T GAT T AC A GGAGAT T Ϊ GGGGAAAAT C GAT GAAC GT GG GT ATGT T CACAT T C T T GGGC G C GGTAAG GAT C T T G T GAT CACC GGT G GC T T TAAC GT C TAT C CAAAAGAAAT T GAAT C AGAGATCGACGCCATGCCAGGGGTAGTGGAATCTGCGGTAATT GGCGTGCCCCATGCGGATTTTGGTGAAGGCGTCACCGCCGTCG T T GT AC GC GAT AAAGGAGC GAC GAT C G AT GAAGC C C AGGT AC T T CAT GGAC T GGAC GGACAGT TAGCCAAGT T TAAGAT GC CGAAG AAGGTAATCTTT'GTGGACGATCTTCCTCGTAACACAATGGGTA AGG'TAC AAAAAAA C GT T C Τ' GC GC GAGA C T T AC AA AG AC AT T TA TAAA

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Claims

Claims

1. A bacterium comprising gene sequence(s) encoding one or more propionate catabolism enzyme(s) operably linked to a directly or indirectly inducible promoter that is not associated with the propionate catabolism enzyme gene in nature.
2. The bacterium of claim 1, wherein the bacterium further comprises gene sequence(s) encoding one or more transporter(s) of propionate operably linked to a promoter that is not associated with the transporter gene in nature.
3. The bacterium of claim 1 or claim 2, wherein the bacterium further comprises gene sequence(s) encoding one or more exporter(s) of succinate operably linked to a promoter that is not associated with the transporter gene in nature.
4. The bacterium of any one of claims 1-3, wherein the bacterium further comprises a genetic modification that reduces the import of succinate into the bacterium.
5. The bacterium any one of claims 2-4, wherein the promoter is a directly or indirectly inducible promoter.
6. The bacterium of any one of claims 1-5, wherein the bacterium further comprises a genetic modification that reduces endogenous biosynthesis of propionate in the bacterium.
7. The bacterium of any of claims 2-6, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme and the promoter operably linked to the gene sequence(s) encoding a transporter of propionate are separate copies of the same promoter.
8. The bacterium of any of claims 2-6, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme and the promoter operably

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9. The bacterium of any of claims 2-6, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme and the promoter operably linked to the gene sequence(s) encoding a transporter of propionate are different promoters.
10. The bacterium of any one of claims 1-9, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.
11. The bacterium of claim any one of claims 1-10, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme is directly or indirectly induced under low-oxygen or anaerobic conditions.
12. The bacterium of claim any one of claims 1-11, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter.
13. The bacterium of claim any one of claims 1-12, wherein the promoter operably linked to the gene sequence(s) encoding a propionate catabolism enzyme is an FNRS promoter.
14. The bacterium of any one of claims 2-13, wherein the promoter operably linked to the gene sequence(s) encoding a transporter of propionate is directly or indirectly induced by exogenous environmental conditions found in the mammalian gut.

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15. The bacterium of claim any one of claims 2-14, wherein the promoter operably linked to the gene sequence(s) encoding a transporter of propionate is directly or indirectly induced under low-oxygen or anaerobic conditions.
16. The bacterium of claim any one of claims 2-15, wherein the promoter operably linked to the gene sequence(s) encoding a transporter of propionate is selected from the group consisting of an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter.
17. The bacterium of any one of claims 1-16, wherein the gene sequence(s) encoding a propionate catabolism enzyme is located on a chromosome in the bacterium.
18. The bacterium of any one of claims 1-17, wherein the gene sequence(s) encoding a propionate catabolism enzyme is located on a plasmid in the bacterium.
19. The bacterium of any one of claims 1-18, wherein the bacterium comprises gene sequence(s) encoding one or more propionate catabolism enzyme(s) that convert propionate to succinate.
20. The bacterium of any one of claims 1-19, wherein the bacterium comprises gene sequence(s) encoding one or more propionate catabolism enzyme(s) selected from prpE, pccB, accAl, mmcE, mutA, and mutB.
21. The bacterium of any one of claims 1-20, wherein the gene sequence(s) encoding one or more propionate catabolism enzyme(s) are present in a single gene cassette.
22. The bacterium of any one of claims 1-21, wherein the bacterium comprises at least two gene sequence(s) encoding one or more propionate catabolism enzyme(s) and wherein the gene sequences are present in two or more separate gene cassettes.

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23. The bacterium of claim 22, wherein the gene sequence(s) encoding one or more propionate catabolism enzyme(s) are present in a first gene cassette, operably linked to a first promoter and present in a second gene cassette, operably linked to a second promoter.
24. The bacterium of claim 23, wherein the first promoter and the second promoter are inducible promoters.
25. The bacterium of claim 23 or claim 24, wherein the first promoter and the second promoter are different promoters.
26. The bacterium of claim 23 or claim 24, wherein the first promoter and the second promoter are separate copies of the same promoter.
27. The bacterium of any of claims 23-26, wherein the first gene cassette comprises prpE, pccB, and accAl and the second gene cassette comprises mmcE, mutA, and mutB.
28. The bacterium of claim 27, wherein the gene sequence(s) encoding prpE has at least 90% identity to SEQ ID NO: 25.
29. The bacterium of claim 27, wherein the gene sequence(s) encoding pccB has at least 90% identity to SEQ ID NO: 39.
30. The bacterium of claim 27, wherein the gene sequence(s) encoding accAl has at least 90% identity to SEQ ID NO: 38.
31. The bacterium of claim 27, wherein the gene sequence(s) encoding mmcE has at least 90% identity to SEQ ID NO: 32.
32. The bacterium of claim 27, wherein the gene sequence(s) encoding mutA has at least 90% identity to SEQ ID NO: 33.
33. The bacterium of claim 27, wherein the gene sequence(s) encoding mutB has at least 90% identity to SEQ ID NO: 34.

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34. The bacterium of any of claims 1-26, wherein the bacterium comprises one or more gene sequence(s) encoding one or more propionate catabolism enzyme(s) that convert propionate to polyhydroxyalkanoate.
35. The bacterium of claim 34, wherein the bacterium comprises one or more gene sequence(s) encoding prpE, phaB, phaC, and phaA.
36. The bacterium of claim 35, wherein the gene sequence(s) encoding prpE has at least 90% identity to SEQ ID NO: 25.
37. The bacterium of claim 35, wherein the gene sequence(s) encoding phaB has at least 90% identity to a sequence encoding SEQ ID NO: 26.
38. The bacterium of claim 35, wherein the gene sequence(s) encoding phaC has at least 90% identity to a sequence encoding SEQ ID NO: 27.
39. The bacterium of claim 35, wherein the gene sequence(s) encoding phaA has at least 90% identity to a sequence encoding SEQ ID NO: 28.
40. The bacterium of any of claims 1-26, wherein the bacterium comprises gene sequence(s) encoding one or more propionate catabolism enzyme(s) that convert propionate to pyruvate and succinate.
41. The bacterium of claim 40, wherein the one or more gene sequence(s) encode prpB, a prpC, and prpD.
42. The bacterium of claim 40 or claim 41, wherein the one or more gene sequence(s) encode prpE.
43. The bacterium of claim 40 or claim 41, wherein the gene sequence(s) encoding prpE has at least 90% identity to SEQ ID NO: 25.

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44. The bacterium of claim 40 or claim 41, wherein the one or more gene sequence(s) encoding prpC has at least 90% identity to SEQ ID NO: 57.
45. The bacterium of claim 40 or claim 41, wherein the one or more gene sequence(s) encoding prpD has at least 90% identity to SEQ ID NO: 58.
46. The bacterium of claim 40 or claim 41, wherein the one or more gene sequence(s) encoding prpB has at least 90% identity to SEQ ID NO: 56.
47. The bacterium of any of claims 1-46 wherein the one or more gene sequence(s) encoding one or more propionate catabolism enzyme(s) comprise one or more gene(s) encoding one or more propionate catabolism enzyme(s) located on a plasmid in the bacterial cell.
48. The bacterium of claims 1-47 wherein the one or more gene sequence(s) encoding one or more propionate catabolism enzyme(s) comprise one or more gene(s) encoding one or more propionate catabolism enzyme(s) located on a chromosome in the bacterial cell.
49. The bacterium of any of claims 3-48, wherein the gene sequence(s) encoding the succinate exporter encodes dcuC.
50. The bacterium of claim 49, wherein the gene sequence(s) encoding dcuC is at least about 90% identity to the sequence of SEQ ID NO: 49.
51. The bacterium of any of claims 3-48, wherein the gene sequence(s) encoding the succinate exporter encodes sucEl.
52. The bacterium of claim 51, wherein the gene sequence(s) encoding sucEl has at least about 90% identity to the sequence of SEQ ID NO: 46.
53. The bacterium of any one of claims 1-52, wherein the engineered bacterial cell further comprises a genetic modification that increases activity of the at least one heterologous gene encoding the at least one propionate catabolism enzyme.

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54. The bacterium of any one of claims 1-53, wherein the engineered bacterial cell further comprises a genetic modification that increases activity of prpE.
55. The bacterium of any one of claims 1-54, wherein the engineered bacterial cell further comprises a genetic modification in pka.
56. The bacterium of any one of claims 1-55, wherein the bacterium is a probiotic bacterial cell.
57. The bacterium of any one of claims 1-56, wherein the bacterium is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus and Lactococcus.
58. The bacterium of any one of claims 1-57, wherein the bacterium is of the genus Escherichia.
59. The bacterium of any one of claims 1-58, wherein the engineered bacterial cell is of the species Escherichia coli strain Nissle.
60. The bacterium of any one of claims 1-59, wherein the engineered bacterial cell is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
61. The bacterium of claim 60, wherein the mammalian gut is a human gut.
62. The bacterium of claim 60 or claim 61, wherein the engineered bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.
63. The bacterium of claims 1-62, wherein the engineered bacterial cell is further engineered to harbor a gene encoding a substance that is toxic to the bacterium, wherein the gene is under the control of a promoter is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut.

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64. A pharmaceutical composition comprising the bacterium in any of claims 1-63, and a pharmaceutically acceptable carrier.
65. The pharmaceutical composition of claim 64 formulated for oral administration.
66. A method for reducing the levels of propionate, methylmalonate and their byproduct molecules in a subject and/or treating a disease or disorder involving the catabolism of propionate in a subject, the method comprising administering a pharmaceutical composition of claim 64 or claim 65.
67. The method of claims 66, wherein the disorder involving the catabolism of propionate is an organic acidemia.
68. The method of claim 67, wherein the organic acidemia is propionic acidemia (PA).
69. The method of claim 67, wherein the organic acidemia is methylmalonic acidemia (MMA).

70. The method of claim 66, wherein the disorder involving the catabolism of propionate is a vitamin Bn deficiency.

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