US 2016O160223A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0160223 A1 Koepke et al. (43) Pub. Date: Jun. 9, 2016

(54) RECOMBINANT MICROORGANISMS CI2N 9/02 (2006.01) EXHIBITING INCREASED FLUX THROUGH CI2N 9/10 (2006.01) A FERMENTATION PATHWAY CI2P 7/18 (2006.01) CI2N 15/52 (2006.01) (71) Applicant: LanzaTech New Zealand Limited, (52) U.S. Cl. Skokie, IL (US) CPC, C12N 15/74 (2013.01); C12P 7/18 (2013.01); CI2N 15/52 (2013.01); C12N 9/0008 (72) Inventors: Michael Koepke, Skokie, IL (US); (2013.01); CI2N 9/1022 (2013.01): CI2N 9/88 Alexander Paul Mueller, Skokie, IL (2013.01); C12Y 102/07001 (2013.01); C12Y (US); Loan Phuong Tran, Skokie, IL 202/01006 (2013.01); C12Y 401/01005 (US) (2013.01) (21) Appl. No.: 14/961,146 (57) ABSTRACT (22) Filed: Dec. 7, 2015 The invention provides a recombinant, carboxydotrophic Related U.S. Application Data Clostridium bacterium that expresses one or more of pyru (60) Provisional application No. 62/168.969, filed on Jun. vate:ferredoxin (EC 1.2.7.1), acetolactate 1, 2015, provisional application No. 62/089,053, filed synthase (EC 2.2.1.6). and acetolactate decarboxylase (EC On Dec. 8, 2014. 4.1.1.5). The invention further provides a method of produc s ing a fermentation product by fermenting the recombinant Publication Classification bacterium in the presence of a gaseous Substrate comprising CO to produce one or more of ethanol, butanol, isopropanol, (51) Int. Cl. isobutanol, higher alcohols, butanediol. 2,3-butanediol. Suc CI2N 15/74 (2006.01) cinate, isoprenoids, fatty acids, biopolymers, and mixtures CI2N 9/88 (2006.01) thereof. Patent Application Publication Jun. 9, 2016 Sheet 1 of 8 US 2016/O160223 A1

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RECOMBINANT MICROORGANISMS ism for 2,3-butanediol formation via pyruvate, which allows EXHIBITING INCREASED FLUX THROUGH for the identification of rate-limiting pathway reactions. A FERMENTATION PATHWAY 0008 FIG. 3 is a diagram showing the 2,3-butanediol pathway and the associated branched-chain bio CROSS REFERENCE TO RELATED synthesis pathway. Pyruvate is converted to O-acetolactate, APPLICATIONS the intermediate for both the 2,3-butanediol and the branched-chain amino acid biosynthesis pathways. Experi 0001. This application claims the benefit of U.S. Patent ments were performed to overexpress PFOR, alsS and alsD. Application 62/089,053 filed Dec. 8, 2014 and U.S. Patent 0009 FIG. 4 is a schematic representation of the Application 62/168,969 filed Jun. 1, 2015, the entirety of pMTL83159 plasmid. The plasmid contains a Gram negative which are incorporated herein by reference. origin of replication (ColE1) , a Gram positive origin of replication (repH) gene, the transfer gene tra.J., the catP gene BACKGROUND encoding for the chloramphenicol/thiamphenicol resistance, 0002 Carboxydotrophic microorganisms may be engi a multiple cloning site locating within lacZ alpha coding neered to produce products, such as fuels and chemicals, sequence and, a ferredoxin gene promoter (Pa.). through fermentation of a gaseous Substrate. Efforts to 0010 FIG. 5 is a set of graphs showing of the growth and improve product concentration and Substrate utilization have metabolite profiles (biomass, 2,3-butanediol (BDO), acetic historically focused on Strain selection and optimization of acid, and ethanol) versus time of five strains grown in Schott fermentation conditions (Abubackar, Bioresour Technol, bottles. 114: 518-522, 2012). The metabolism of natural microorgan 0011 FIG. 6 is a set of graphs showing the metabolite isms, however, did not evolve to achieve commercial objec profile (top graphs) and gas profile (bottom graphs) of the tives of high yields, rates, and titers, such that certain com combined PFOR, alsS and alsDoverexpression strain and the mercial objectives cannot be achieved through mere strain plasmid control strain at a 4 mol/L/d CO uptake over the selection and optimization of fermentation conditions. course of 20 days. Accordingly, there remains a need for improved microorgan 0012 FIG. 7 is a set of graphs showing the metabolite and isms and methods for the production of useful products. Such the gas profiles of the overexpression culture at an 8 mol/L/d as fuels and chemicals. CO uptake over the course of 11 days. 0013 FIG. 8 is a set of graphs showing biomass and SUMMARY OF THE INVENTION butanediol production of cultures expressing A. hydrophila alsD (open squares), L. lactis alsD (closed squares), and 0003. The invention provides a recombinant, carboxy empty plasmid control (triangles). Values are the average of dotrophic Clostridium bacterium comprising one or more three replicates and error bars represent one standard devia selected from the group consisting of pyruvate: tion. ferredoxin oxidoreductase (EC 1.2.7.1), acetolactate syn thase (EC 2.2.1.6), and acetolactate decarboxylase (EC 4.1. DETAILED DESCRIPTION OF THE INVENTION 1.5), wherein each is an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous 0014. A fermentation pathway is a cascade of biochemical enzyme. The recombinant bacterium may express one, two, reactions (pathway reactions) by which a substrate, prefer or all three of these enzymes. ably a gaseous Substrate, is converted to a fermentation prod 0004. The recombinant bacterium may be derived from uct. Pathway reactions typically involve enzymes that catal any Clostridium microorganism. In one embodiment, the yse or increase the rate of the pathway reaction. recombinant bacterium is derived from C. autoethanogenium, (0015 “Flux” refers to the flow of metabolites through one C. liungdahli, or C. ragsdalei. In a preferred embodiment, or more reactions in a fermentation pathway. The flux through the recombinant bacterium is derived from C. autoethanoge individual pathway reactions has an upper and lower limit. num deposited under DSMZ Accession No. DSM23693 (C. Therefore, the flux may be changed by adjusting conditions or autoethanogenium LZ1561). factors that affect enzymatic activity. Adjustment of the flux 0005. The invention further provides a method of produc through one pathway reaction may alter the overall flux of the ing a fermentation product, comprising fermenting the fermentation pathway. Flux may be measured according to recombinant bacterium in the presence of a gaseous Substrate any method known in the art. By way of example, flux may be comprising CO to produce one or more of ethanol, butanol, measured using flux-balance analysis (FBA) (Gianchandani, isopropanol, isobutanol, higher alcohols, butanediol. 2,3-bu Systems Biol Medicine, 2:372-382, 2010). Flux through the tanediol. Succinate, isoprenoids, fatty acids, biopolymers, pathway may also be measured by the level of metabolites and and mixtures thereof. products (metabolomics) (Patti, Nat Rev Molec Cell Biol, 13: 263-269, 2012) and/or labelling experiments as C13 (flux BRIEF DESCRIPTION OF THE DRAWINGS omics) (Niittylae, Methods Mol Biol, 553: 355-372, 2009: Tang, Mass Spectrom Rev. 28:362-375, 2009). 0006 FIG. 1 is a flux map of the ethanol biosynthesis 0016. The efficiency of a fermentation pathway can be pathway detailing the measured enzyme activities and flux increased by increasing the reaction flux through the pathway. through a carboxydotrophic microorganism for ethanol for The increased flux results in one or more of an increased rate mation via acetyl-CoA, which allows for the identification of of growth of microorganisms performing the fermentation, an rate-limiting pathway reactions. The thickness of the arrows increased rate of growth and/or product production rate at is proportional to the activity of the particular pathway reac elevated product concentrations, an increased fermentation tion. product concentration in the fermentation broth, an increased 0007 FIG. 2 is a flux map detailing the measured enzyme volume of fermentation product produced per volume of sub activities and flux through a carboxydotrophic microorgan strate consumed, an increased rate of production or level of US 2016/0160223 A1 Jun. 9, 2016 production of the fermentation product. Preferably, the to a parental microorganism. Genetic modification includes increased efficiency results in an increased fermentation insertion, deletion, or Substitution of nucleic acids, for product production rate. example. 0017. One method to identify rate limiting reactions 0023. In general, the term “derived from indicates that a (bottlenecks) is to measure enzyme activities for all reactions nucleic acid, , or microorganism is modified or involved in the fermentation pathway from substrate to prod adapted from a different (i.e., a parental or wild-type) nucleic uct. This can be done by analysing the enzymatic activity of acid, protein, or microorganism, respectively, so as to pro reactions in cells growing under process conditions to iden duce a new recombinant microorganism. tify the reactions with the lowest rates. These can then be 0024 Methods of genetic modification of a parental adjusted so as not to be rate limiting, thus increasing the flux microorganism include molecular methods such as heterolo throughout the system. Enzymatic activity may be measured gous gene expression, genome insertion or deletion, altered by any method known in the art, such as the methods gene expression or inactivation of , or enzyme engineer described in Huang, J Bacteriol, 194; 3689-3699, 2012. ing methods. Such techniques are described, for example, in Sambrook, Molecular Cloning: A Laboratory Manual, Cold 0018. The inventors have analysed the activity of enzymes Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., involved infermentation pathways and found that some path 2001: Pleiss, Curr Opin Biotechnol, 22: 611-617, 2011; Park, way reactions exhibit substantially lower enzymatic activity Protein Engineering and Design, CRC Press, 2010. Expres than other reactions in the same pathway. The recombinant sion constructs/vectors may contain, for example, one or microorganisms and methods described herein have particu more promoters or ribosomal binding sites. Nucleic acids and lar utility for pathways where the product yield in a parental construct/vector sequences described herein may also contain microorganism may lack the product yield to be a viable standard linker nucleotides such as those required for ribo commercial target. Some binding sites and/or restriction sites. 0019. Examples of fermentation pathways that are ame 0025 Nucleic acids and nucleic acid constructs, including nable to analysis of enzyme activity include the Wood expression constructs/vectors of the invention may be con Ljungdahl pathway, fermentation pathways to produce etha structed using any method known in the art. For example, nol. 2,3-butanediolora precursor thereof such as acetyl-CoA chemical synthesis or recombinant techniques may be used. and pyruvate, and biosynthesis pathways for cofactors tet Such techniques are described, for example, in Sambrook, rahydrofolate and cobalamine (B) which may be required in Molecular Cloning: A Laboratory Manual, Cold Spring Har fermentation pathways. The Wood-Ljungdahl pathway is bor Laboratory Press, Cold Spring Harbor, N. Y., 1989. composed of a number of reactions catalysed by enzymes, as Essentially, the individual genes and regulatory elements may described in FIG. 1 and FIG. 2. The steps subsequent to the be operably linked to one another Such that the genes can be Wood-Ljungdahl pathway which lead to the production of expressed to form the desired . Suitable vectors will desirable fermentation products are also considered to be part be appreciated by those of ordinary skill in the art. However, of the fermentation pathway. In a particular embodiment, the by way of example, the following vectors may be suitable: fermentation pathway results in the production of a fermen pMTL80000 vectors, pIMP1, p.JIR750, and other plasmids. tation product selected from the group consisting of ethanol, 0026. Nucleic acids may be delivered to a microorganism butanol, isopropanol, isobutanol, higher alcohols, butanediol. using any method known in the art. For example, nucleic 2,3-butanediol. Succinate, isoprenoids, fatty acids, biopoly acids may be delivered to a microorganism as naked nucleic mers, and mixtures thereof. acids or may be formulated with one or more agents (e.g., 0020. The invention provides a recombinant, carboxy liposomes) to facilitate the transformation process to the dotrophic Clostridium bacterium comprising one or more microorganism. The nucleic acids may be DNA, RNA, enzymes selected from the group consisting of pyruvate: cDNA, or combinations thereof, as is appropriate. Restriction ferredoxin oxidoreductase (EC 1.2.7.1), acetolactate syn inhibitors may be used in certain embodiments (Murray, thase (EC 2.2.1.6), and acetolactate decarboxylase (EC 4.1. Microbiol Molec Biol Rev. 64: 412-434, 2000). Additional 1.5), wherein each enzyme is an overexpressed endogenous vectors include include plasmids, viruses (including bacte enzyme, a mutated endogenous enzyme, or an exogenous riophage), , and artificial . In a pre ferred embodiment, nucleic acids are delivered to a microor enzyme. ganism using a plasmid. By way of example only, 0021 A parental microorganism' is a microorganism transformation (including transduction or transfection) may used to generate a recombinant microorganism of the inven beachieved by electroporation, ultrasonication, polyethylene tion. The parental microorganism may be one that occurs in glycol-mediated transformation, chemical or natural compe nature (i.e., a wild type microorganism) or one that has been tence, protoplast transformation, prophage induction or con previously modified (i.e. a recombinant microorganism). The jugation. Suitable transformation techniques are described recombinant microorganisms of the invention may be modi for example in, Sambrook, Molecular Cloning: A Laboratory fied to express or overexpress one or more enzymes that were Manual, Cold Spring Harbor Laboratory Press, Cold Spring or were not expressed or overexpressed in the parental micro Harbor, N.Y., 1989. organism, or may be modified to exhibit increased availability 0027. The use of electroporation has been reported for of one or more co-factors. In one embodiment, the parental several carboxydotrophic acetogens, including C. liungdahli organism may be C. autoethanogenium, C. ljungdahli, or C. (Koepke, PNAS, 107: 13087-13092, 2010; WO/2012/ ragsdalei. In a particularly preferred embodiment, the paren 053905), C. autoethanogenium (WO/2012/053905), C. aceti tal organism is C. autoethanogenium LZ1561, which is depos cum (Schiel-Bengelsdorf, Synthetic Biol, 15: 2191-2198. ited under DSMZ accession DSM23693. 2012), and A. woodii (Strátz, Appl Environ Microbiol, 60: 0022. A “recombinant microorganism' is a microorgan 1033-1037, 1994). The use of electroporation has also been ism that has undergone genetic modification when compared reported in Clostridia, including C. acetobutylicum (Mermel US 2016/0160223 A1 Jun. 9, 2016

stein, Biotechnol, 10:190-195, 1992), and C. cellulolyticum 2008/028055). Indole production was observed with all three (Jennert, Microbiol, 146: 3071-3080, 2000). Additionally, species as well. However, the species differentiate in sub prophage induction has been demonstrated for carboxy strate utilization of various Sugars (e.g., rhamnose, arabi dotrophic acetogens, including C. Scatologenes (Parthasar nose), acids (e.g., gluconate, citrate), amino acids (e.g., argi athy, Development of a Genetic Modification System in nine, histidine), or other Substrates (e.g., betaine, butanol). Clostridium scatologenes ATCC 25775 for Generation of Moreover some of the species were found to be auxotrophic Mutants, Masters Project, Western Kentucky University, to certain vitamins (e.g., thiamine, biotin) while others were 2010), and conjugation has been described for many not. The organization and number of Wood-Ljungdahl path Clostridia, including C. difficile (Herbert, FEMS Microbiol way genes, responsible for gas uptake, has been found to be Lett, 229: 103-1 10, 2003) and C. acetobuylicum (Williams, J the same in all species, despite differences in nucleic and Gen Microbiol, 136: 819-826, 1990). Similar methods could amino acid sequences (Köpke, Curr Opin Biotechnol, 22: be used in carboxydotrophic acetogens. 320-325, 2011). Also, reduction of carboxylic acids into their 0028. The invention provides a recombinant carboxy corresponding alcohols has been shown in a range of these dotrophic Clostridium bacterium adapted to exhibit increased microorganisms (Perez, Biotechnol Bioeng, 110:1066-1077, flux through a fermentation pathway relative to a parental 2012). These traits are therefore not specific to one organism microorganism. In one particular embodiment of the inven like C. autoethanogenium or C. liungdahli, but rather general tion, the parental microorganism is selected from the group of traits for carboxydotrophic, ethanol-synthesizing Clostridia carboxydotrophic Clostridia comprising C. autoethanoge and it can be anticipated that mechanisms work similarly num, C. liungdahli, C. ragsdalei, C. carboxidivorans, C. across these strains, although there may be differences in drakei, C. Scatologenes, C. aceticum, C. formicoaceticum, performance. and C. magnum. 0031. In one embodiment, the parental microorganism is 0029. The recombinant bacterium may be derived from C. autoethanogenium, C. ljungdahli, or C. ragsdalei. Prefer the cluster of carboxydotrophic Clostridia comprising the ably, the parental microorganism is wild-type C. autoethano species C. autoethanogenium, C. liungdahli, C. ragsdalei, genum or C. autoethanogenium deposited under DSMZacces and related isolates. These include but are not limited to sion number DSM100.61 or DSM23693 (C. strains C. autoethanogenium JAI-1T (DSM100.61) (Abrini, autoethanogenium LZ1561). In one embodiment, the recom Arch Microbiol, 161: 345-351, 1994), C. autoethanogenium binant bacterium is derived from C. autoethanogenium, C. LBS1560 (DSM19630) (WO 2009/064200), C. autoethano liungdahlii, or C. ragsdalei. Preferably, the recombinant bac genum LZ1561 (DSM23693), C. Jjungdahlii PETCT terium is derived from wild-type C. autoethanogenium or C. (DSM13528=ATCC 55383) (Tanner, IntJSyst Bacteriol, 43: autoethanogenium deposited under DSMZ accession number 232-236, 1993), C. ljungdahlii ERI-2 (ATCC 55380) (U.S. DSM23693 (C. autoethanogenium LZ1561). Pat. No. 5,593,886), C. ljungdahlii C-01 (ATCC 55.988) (U.S. 0032. The enzymes and genes of the invention may be Pat. No. 6,368.819), C. ljungdahliiO-52 (ATCC 55989) (U.S. overexpressed endogenous enzymes and genes, mutated Pat. No. 6,368.819), C. ragsdalei P11T (ATCC BAA-622) endogenous enzymes and genes, or exogenous enzymes and (WO 2008/028055), related isolates such as “C. coskatii” genes. (U.S. Publication 2011/0229947), or mutated strains such as 0033 “Endogenous” refers to a nucleic acid or protein that C. liungdahlii OTA-1 (Tirado-Acevedo, Production of Bioet is present in the wild-type or parental bacterium from which hanol from Synthesis Gas Using Clostridium ljungdahli, the recombinant bacterium of the invention is derived. In one PhD thesis, North Carolina State University, 2010). These embodiment, the expression of an endogenous gene may be strains form a subcluster within the Clostridial rRNA cluster controlled by an exogenous regulatory element, such as an I, and their 16S rRNA gene is more than 99% identical with a exogenous promoter. similarlow GC content of around 30%. However, DNA-DNA reassociation and DNA fingerprinting experiments showed 0034) “Exogenous” refers to a nucleic acid or protein that that these strains belong to distinct species (WO 2008/ is not present in the wild-type or parental bacterium from 028055). The strains of this cluster are defined by common which the recombinant bacterium of the invention is derived. characteristics, having both a similar genotype and pheno In one embodiment, an exogenous gene or enzyme may be type, and they all share the same mode of energy conservation derived from a heterologous strain or species and introduced to or expressed in the recombinant bacterium. In another and fermentative metabolism. The strains of this cluster lack embodiment, an exogenous gene or enzyme may be artifi cytochromes and conserve energy via an Rnf complex. cially or recombinantly created. Exogenous nucleic acids 0030 All species of the above-referenced cluster have a may be adapted to integrate into the genome of the bacterium similar morphology and size (logarithmic growing cells are or to remain in an extra-chromosomal state in the bacterium, between 0.5-0.7x3-5 um), are mesophilic (optimal growth for example, in a plasmid. temperature between 30–37° C.), and are strictly anaerobic (Abrini, Arch Microbiol, 161: 345-351, 1994; Tanner, Int J 0035 “Enzyme activity” refers broadly to enzymatic Syst Bacteriol, 43: 232-236, 1993; and WO 2008/028055). activity, including, but not limited, to the activity of an Moreover, they all share the same major phylogenetic traits, enzyme, the amount of an enzyme, or the availability of an Such as same pH range (pH 4-7.5, with an optimal initial pH enzyme to catalyse a reaction. Accordingly, “increasing of 5.5-6), strong autotrophic growth on CO-containing gases enzyme activity includes an increase in the activity of an with similar growth rates, and a similar metabolic profile with enzyme, an increase in the amount of an enzyme, or an ethanol and acetic acid as main fermentation end products, increase in the availability of an enzyme to catalyse a reac and Small amounts of 2,3-butanediol and lactic acid formed tion. under certain conditions (Abrini, Arch Microbiol, 161: 345 0036. The genes and enzymes of the invention may be 351, 1994; Köpke, Curr Opin Biotechnol, 22:320-325, 2011; developed or engineered using any method known in the art, Tanner, Int J Syst Bacteriol, 43: 232-236, 1993; and WO including, for example, directed evolution, knowledge-based US 2016/0160223 A1 Jun. 9, 2016

design, random mutagenesis methods, gene shuffling, codon form substantially the same function. For nucleic acid optimization, use of site-specific libraries, and use of site sequences that encode a protein or peptide this means that the evaluation libraries. encoded protein or peptide has substantially the same func 0037 “Mutated’ refers to a nucleic acid or protein that has tion. For nucleic acid sequences that represent promoter been modified in the recombinant bacterium of the invention sequences, the variant sequence will have a similar ability to compared to the wild-type or parental bacterium from which promote expression of one or more genes. Such nucleic acids the recombinant bacterium of the invention is derived. In one or proteins may be referred to herein as “functionally equiva embodiment, the may be a deletion, insertion, or lent variants. By way of example, functionally equivalent Substitution in a gene encoding an enzyme. In another variants of a nucleic acid include allelic variants, fragments of embodiment, the mutation may be a deletion, insertion, or a gene, genes which include (deletion, insertion, Substitution of one or more amino acids in an enzyme. nucleotide Substitutions and the like) and/or polymorphisms 0038 “Codon optimization” refers to the mutation of a and the like. Homologous genes from other microorganisms nucleic acid, Such as a gene, for optimized or improved trans may also be considered examples of functionally equivalent lation of the nucleic acid in a particular strain or species. variants of the sequences specifically exemplified herein. Codon optimization may result in faster translation rates or These include homologous genes in species such as C. aceto higher translation accuracy. In a preferred embodiment, the butyllicum, C. beijerinckii, or C. Jiungdahlii, the details of genes encoding the enzymes of the invention are codon opti which are publicly available on websites such as Genbank or mized for expression in Clostridium, particularly C. autoet NCBI. Functionally equivalent variants also includes nucleic hanogenium, C. liungdahli, and/or C. ragsdalei. In a further acids whose sequence varies as a result of codon optimization preferred embodiment, the genes encoding the enzymes of for a particular organism. A functionally equivalent variant of the invention are codon optimized for expression in C. auto a nucleic acid will preferably have at least approximately ethanogenium LZ1561. 70%, approximately 80%, approximately 85%, approxi 0039 “Overexpressed’ refers to any increase in expres mately 90%, approximately 95%, approximately 98%, or sion of a nucleic acid or protein in the recombinant bacterium greater nucleic acid sequence identity with the specified of the invention compared to the wild-type or parental bacte nucleic acid. A functionally equivalent variant of a protein rium from which the recombinant bacterium of the invention will preferably have at least approximately 70%, approxi is derived. Overexpression may be achieved by any means mately 80%, approximately 85%, approximately 90%, known in the art, including modifying gene copy number, approximately 95%, approximately 98%, or greater amino gene rate, gene translation rate, or enzyme deg acid identity with the specified protein. Such variants include radation rate. a fragment of a protein or peptide wherein the fragment 0040 “Overexpressed endogenous enzyme” refers to an comprises a truncated form of the protein or peptide wherein endogenous enzyme that is present at higher levels in the deletions may be from 1 to 5, to 10, to 15, to 20, to 25 amino recombinant bacterium of the invention compared to the acids, and may extend from residue 1 through 25 at either wild-type or parental bacterium from which the recombinant terminus of the polypeptide, and wherein deletions may be of bacterium of the invention is derived. The overexpressed any length within the region or may be at an internal location. endogenous enzyme may likewise be encoded by an endog The functional equivalence of a variant nucleic acid or protein enous gene, which may be modified, for example, to be con may be evaluated using any method known in the art. How trolled by a strong or constitutive promoter. Similarly, “over ever, by way of example, assays to test for the activity of expressed endogenous gene’ refers to an endogenous gene certain enzymes are described in Huang, J Bacteriol, 194: that is present or transcripted at higher rates or levels in the 3689-3699, 2012. recombinant bacterium of the invention compared to the 0044. In certain embodiments having active restriction wild-type or parental bacterium from which the recombinant enzyme systems, it may be necessary to methylate a nucleic bacterium of the invention is derived. acid before introduction of the nucleic acid into a microor 0041. “Mutated endogenous enzyme” refers to an endog ganism. enous enzyme that is mutated or modified in the recombinant 0045 Generally, methylation is performed using a shuttle bacterium of the invention compared to the wild-type or microorganism, preferably a restriction negative shuttle parental bacterium from which the recombinant bacterium of microorganism, Such as E. coli, B. subtilis, or L. lactis, that the invention is derived. Similarly, “mutated endogenous facilitates the methylation of the nucleic acid sequences that gene' refers to an endogenous gene that is mutated or modi make up the expression construct/vector. The methylation fied in the recombinant bacterium of the invention compared construct/vector comprises a nucleic acid sequence encoding to the wild-type or parental bacterium from which the recom a methyltransferase. Once the expression construct/vector binant bacterium of the invention is derived. and the methylation construct/vector are introduced into the 0042 “Exogenous enzyme” refers to an enzyme that is not shuttle microorganism, the methyltransferase gene present on present in the wild-type or parental bacterium from which the the methylation construct/vector is induced. Induction may recombinant bacterium of the invention is derived. Similarly, be by any suitable promoter system although in one particular “exogenous gene' refers to a gene that is not present in the embodiment of the invention, the methylation construct/vec wild-type or parental bacterium from which the recombinant tor comprises an inducible lac promoter and is induced by bacterium of the invention is derived. Typically, the exog addition of lactose or an analogue thereof, more preferably enous enzyme or gene is derived from a heterologous strain or isopropyl-B-D-thio-galactoside (IPTG). Other suitable pro species and introduced to or expressed in the recombinant moters include the ara, tet, or T7 system. In a further embodi bacterium. ment, the methylation construct/vector promoter is a consti 0043. The invention may be practiced using variant tutive promoter. nucleic acids or proteins whose sequence varies from the 0046. In a particular embodiment, the methylation con sequences specifically exemplified herein provided they per struct/vector has an origin of replication specific to the iden US 2016/0160223 A1 Jun. 9, 2016

tity of the shuttle microorganism so that any genes present on one molecule (the reductant, or electron donor) to another the methylation construct/vector are expressed in the shuttle (the oxidant, or electron acceptor). Specifically, pyruvate: microorganism. Preferably, the expression construct/vector ferredoxin oxidoreductase catalyses the interconversion of has an origin of replication specific to the identity of the pyruvate and acetyl-CoA: pyruvate+CoA-2 oxidized ferre destination microorganism so that any genes present on the doxin ( ) acetyl-CoA+CO+2 reduced ferredoxin-2 H. expression construct/vector are expressed in the destination Conversion of acetyl-CoA to pyruvate links the Wood microorganism. Ljungdahl pathway of autotrophic CO(2) fixation to the 0047 Expression of the methyltransferase enzyme results reductive tricarboxylic acid cycle, which in autotrophic in methylation of the genes present on the expression con anaerobes is the stage for biosynthesis of all cellular macro struct/vector. The expression construct/vector may then be molecules (Furdi, J Biol Chem, 15: 28494-28499, 2000). isolated from the shuttle microorganism according to any Pyruvate:ferredoxin oxidoreductase may also be known as method known in the art. In one embodiment, both construct/ pyruvate:ferredoxin 2-oxidoreductase (CoA-acetylating), vector are concurrently isolated. The expression construct/ pyruvate oxidoreductase, pyruvate synthase, pyruvate Syn vector may be introduced into the destination microorganism thetase, or pyruvic-ferredoxin oxidoreductase. using any method known in the art. Since the expression 0053. The pyruvate:ferredoxin oxidoreductase enzyme of construct/vector is methylated, the nucleic acid sequences the invention may be an overexpressed endogenous enzyme, present on the expression construct/vector are able to be a mutated endogenous enzyme, or an exogenous enzyme. incorporated into the destination microorganism and be suc Similarly, the pyruvate:ferredoxin oxidoreductase enzyme of cessfully expressed. the invention may be encoded by an endogenous pyruvate: 0048. A methyltransferase gene may be introduced into a ferredoxin oxidoreductase gene that has been engineered for shuttle microorganism and overexpressed. Thus, in one overexpression, may be encoded by a mutated endogenous embodiment, the resulting methyltransferase enzyme may be pyruvate:ferredoxin oxidoreductase gene, or may be encoded collected using known methods and used in vitro to methylate by an exogenous pyruvate:ferredoxin oxidoreductase gene. an expression plasmid. The expression construct/vector may In a preferred embodiment, the pyruvate:ferredoxin oxi then be introduced into the destination microorganism for doreductase enzyme is overexpressed endogenous pyruvate: expression. In another embodiment, the methyltransferase ferredoxin oxidoreductase, Such as overexpressed endog gene is introduced into the genome of the shuttle microorgan enous C. autoethanogenium, C. liungdahli, or C. ragsdalei ism followed by introduction of the expression construct/ pyruvate:ferredoxin oxidoreductase. Pyruvate:ferredoxin vector into the shuttle microorganism, isolation of one or oxidoreductase enzymes are often unstable in the presence of more constructs/vectors from the shuttle microorganism and oxygen. In a preferred embodiment, the pyruvate:ferredoxin then introduction of the expression construct/vector into the oxidoreductase enzyme is oxygen stable or demonstrates at destination microorganism. least Some degree of oxygen insensitivity. In a further pre 0049. The expression construct/vector and the methyla ferred embodiment, the pyruvate:ferredoxin oxidoreductase tion construct/vector may be combined to provide a compo enzyme is exogenous Desulfovibrio africanus pyr/uvate: sition of matter. Such a composition has particular utility in ferredoxin oxidoreductase, or an enzyme derived therefrom. circumventing restriction barrier mechanisms to produce the Expression of D. africanus pyruvate:ferredoxin oxidoreduc recombinant microorganisms of the invention. In one particu tase has been demonstrated in E. coli (Pieulle, J Bacteriol, lar embodiment, the expression construct/vector and/or the 179:5684-5692, 1997), but not in a Clostridium microorgan methylation construct/vector are plasmids. A number of suit 1S able methyltransferases may be used, including, for example, 0054 Acetolactate synthase (Als) (EC 2.2.1.6) is an B. subtilis phage doTI methyltransferase or the methyltrans enzyme that catalyses the first step in the synthesis of ferase described in WO 2012/053905. Similarly, a number of branched-chain amino acids, such as , leucine, and constructs/vectors adapted to allow expression of a methyl . In particular, acetolactate synthase is a transketo transferase gene may be used to generate the methylation lase that has both catabolic and anabolic forms, and catalyses construct/vector. the conversion of two pyruvate molecules to an acetolactate 0050. By way of example, in one embodiment, a recom molecule and carbon dioxide: 2 CH-COCOO binant microorganism of the invention may be produced by a ( ). CHCOCOHCHCOO.--CO. Acetolactate synthase method comprising (a) introduction into a shuttle microor may also be known as acetohydroxy acid synthase. ganism of (i) of an expression construct/vector comprising a 0055. The acetolactate synthase enzyme of the invention nucleic acid as described herein and (ii) a methylation con may be an overexpressed endogenous enzyme, a mutated struct/vector comprising a methyltransferase gene; and (b) endogenous enzyme, or an exogenous enzyme. Similarly, the expression of the methyltransferase gene; isolation of one or acetolactate synthase enzyme of the invention may be more constructs/vectors from the shuttle microorganism; and encoded by an endogenous acetolactate synthase gene that introduction of the one or more construct/vector into a desti has been engineered for overexpression, may be encoded by nation microorganism. In one embodiment, the methyltrans a mutated endogenous acetolactate synthase gene, or may be ferase gene of step (b) is expressed constitutively. In another encoded by an exogenous acetolactate synthase gene. The embodiment, expression of the methyltransferase gene of acetolactate synthase may be anabolic or catabolic. In a pre step (b) is induced. ferred embodiment, the acetolactate synthase enzyme is over 0051. The recombinant bacterium of the invention com expressed endogenous acetolactate synthase, Such as overex prises one or more of pyruvate:ferredoxin oxidoreductase, pressed endogenous C. autoethanogenium, C. liungdahli, or acetolactate synthase, and acetolactate decarboxylase. C. ragsdaleiacetolactate synthase. In particular, the acetolac 0052 Pyruvate:ferredoxin oxidoreductase (PFOR or tate synthase enzyme may be overexpressed endogenous POR) (EC 1.2.7.1) is an enzyme belonging to a family of IlvB, ILVE ORF2059, IlvB ORF2336, IlvC, IlvN, IlvBN, or that catalyses the transfer of electrons from AlsS acetolactate synthase. In a preferred embodiment, the US 2016/0160223 A1 Jun. 9, 2016

acetolactate synthase enzyme is mutated endogenous aceto -continued lactate synthase, such as mutated acetolactate synthase derived from any endogenous C. autoethanogenium, C. SEQ liungdahli, or C. ragsdalei acetolactate synthase. In particu ID NO: Description lar, the mutated endogenous acetolactate synthase may be 3 codon-optimized pyruvate:ferredoxin feedback-insensitive IlvN acetolactate synthase. In a pre oxidoreductase with Xbaland NheI, ferred embodiment, the acetolactate synthase enzyme is C. autoethanogentin LZ1561, nucleic acid sequence 4 codon-optimized pyruvate:ferredoxin oxidoreductase, exogenous acetolactate synthase, such as Bacillus subtilis C. autoethanogentin LZ1561, amino acid sequence acetolactate synthesis, particularly feedback-insensitive B. 5 pyruvate:ferredoxin oxidoreductase with Xbaland subtilis AlsS acetolactate synthase. The expression of B. sub Nehl, D. aficantis, nucleic acid sequence tilis AlsS has been shown in Synechococcus elongatus sp. 6 pyruvate:ferredoxin oxidoreductase, D. africants, amino acid sequence strain PCC 7942 (Oliver, Metabol Eng, 22:76-82, 2014), but 7 native IlvB ORF2059 acetolactate synthase, not in a Clostridium microorganism. C. autoethanogentin LZ1561, nucleic acid sequence 0056 Acetolactate decarboxylase (EC 4.1.1.5) is an 8 native IlvB ORF2059 acetolactate synthase, enzyme belonging to a family of , specifically the car C. autoethanogentin LZ1561, amino acid sequence 9 native IlvB ORF2336 acetolactate synthase, boxy-lyases, which cleave carbon-carbon bonds. Acetolac C. autoethanogentin LZ1561, nucleic acid sequence tate decarboxylase catalyses the reaction of (S)-2-hydroxy O native IlvB ORF2336 acetolactate synthase, 2-methyl-3-oxobutanoate to (R)-2-acetoin and CO: (S)-2- C. autoethanogentin LZ1561, amino acid sequence hydroxy-2-methyl-3-oxobutanoate ( ) (R)-2-acetoin-i-CO. 1 native IlvN acetolactate synthase (regulatory subunit) with NdeI and SacI, Acetolactate decarboxylase may also be known as alpha C. autoethanogentin LZ1561, nucleic acid sequence acetolactate decarboxylase or (S)-2-hydroxy-2-methyl-3-ox 2 native IlvN acetolactate synthase (regulatory subunit), obutanoate carboxy-. C. autoethanogentin LZ1561, amino acid sequence 0057 The acetolactate decarboxylase enzyme of the 3 mutant IlvN (G-10-D) acetolactate synthase (regulatory subunit) with NdeI invention may be an overexpressed endogenous enzyme, a and SacI, C. autoethanogent in LZ1561, nucleic acid sequence mutated endogenous enzyme, or an exogenous enzyme. 4 mutant IlvN (G-10-D) acetolactate synthase (regulatory subunit), Similarly, the acetolactate decarboxylase enzyme of the C. autoethanogentin LZ1561, amino acid sequence invention may be encoded by an endogenous acetolactate 5 native Alss acetolactate synthase, decarboxylase gene that has been engineered for overexpres C. autoethanogentin LZ1561, nucleic acid sequence 6 native Alss acetolactate synthase, Sion, may be encoded by a mutated endogenous acetolactate C. autoethanogenium LZ1561, amino acid sequence decarboxylase gene, or may be encoded by an exogenous 7 codon-optimized AlsS acetolactate synthase with NdeI and SacI, acetolactate decarboxylase gene. In a preferred embodiment, C. autoethanogentin LZ1561, nucleic acid sequence 8 codon-optimized Alss acetolactate synthase, the acetolactate decarboxylase enzyme is overexpressed C. autoethanogentin LZ1561, amino acid sequence endogenous acetolactate decarboxylase, such as overex 9 acetolactate synthase with NdeI and SacI, pressed endogenous C. autoethanogenium, C. liungdahli, or B. subtilis, nucleic acid sequence C. ragsdalei acetolactate decarboxylase. The overexpressed 2O acetolactate synthase, B. subtilis, amino acid sequence 21 native acetolactate decarboxylase, endogenous acetolactate decarboxylase may be BudA aceto C. autoethanogentin LZ1561, nucleic acid sequence lactate decarboxylase or AlsDacetolactate decarboxylase. In 22 native acetolactate decarboxylase, a preferred embodiment, the acetolactate decarboxylase C. autoethanogentin LZ1561, amino acid sequence enzyme is exogenous acetolactate decarboxylase, such as 23 codon-optimized acetolactate decarboxylase with SacI and KpnI, Aeromonas hydrophila acetolactate decarboxylase or Leu C. autoethanogentin LZ1561, nucleic acid sequence conostoc lactis acetolactate decarboxylase. The expression of 24 codon-optimized acetolactate decarboxylase, B. subtilis AlsD has been shown in Synechococcus elongatus C. autoethanogentin LZ1561, amino acid sequence 25 acetolactate decarboxylase, A. hydrophia, nucleic acid sequence sp. strain PCC 7942 (Oliver, Metabol Eng, 22:76-82, 2014), 26 acetolactate decarboxylase, A. hydrophia, amino acid sequence but not in a Clostridium microorganism. 27 acetolactate decarboxylase with SacI and Kipn, 0058. The pyruvate:ferredoxin oxidoreductase, acetolac L. lactis, nucleic acid sequence tate synthase, and acetolactate dehydrogenase enzymes may 28 acetolactate decarboxylase, L. lactis, amino acid sequence comprise or may be derived from any of the amino acid sequences in the following table. Similarly, the genes encod 0059. The recombinant bacterium of the invention may ing the pyruvate:ferredoxin oxidoreductase, acetolactate Syn also comprise any combination of pyruvate:ferredoxin oxi thase, and acetolactate dehydrogenase enzymes may com doreductase, acetolactate synthase, and acetolactate decar prise or may be derived from any of the nucleic acid boxylase. The bacterium may comprise pyruvate:ferredoxin sequences in the following table. Moreover, any of the oxidoreductase and acetolactate synthase, but not acetolac enzymes or genes may be variants of the sequences in the tate decarboxylase. The bacterium may comprise pyruvate: following table. For example, the enzymes or genes may have ferredoxin oxidoreductase and acetolactate decarboxylase, about 80%, about 90%, about 95%, or about 99% sequence but not acetolactate synthase. The bacterium may comprise identity to the sequences in the following table. acetolactate synthase and acetolactate decarboxylase, but not pyruvate:ferredoxin oxidoreductase. Finally, the bacterium SEQ may comprise each of pyruvate:ferredoxin oxidoreductase, ID NO: Description acetolactate synthase, and acetolactate decarboxylase. 1 native pyruvate:ferredoxin oxidoreductase, 0060. The recombinant bacterium of the invention may C. attoethanogentin LZ1561, nucleic acid sequence further express or be engineered to express or overexpress one 2 native pyruvate:ferredoxin oxidoreductase, or more of alcohol dehydrogenase (EC 1.1.1.1), aldehyde C. attoethanogentin LZ1561, amino acid sequence dehydrogenase (acylating) (EC 1.2.1.10), formate dehydro genase (EC 1.2.1.2), formyl-THF synthetase (EC 6.3.2.17), US 2016/0160223 A1 Jun. 9, 2016 methylene-THF dehydrogenase/formyl-THF cyclohydrolase (EC 3.7.1.12), cobalt-precorrin-5B (C1)-methyl (EC:6.3.4.3), methylene-THF reductase (EC 1.1.1.58), CO transferase (EC 2.1.1.195), cobalt-precorrin-7 (C15)-methyl dehydrogenase/acetyl-CoA synthase (EC 2.3.1.169), alde transferase (EC 2.1.1.196), cobaltochelatase CobN (EC 6.6. hyde ferredoxin oxidoreductase (EC 1.2.7.5), phosphotrans 1.2), cobyrinic acid a,c-diamide synthase (EC 6.3.5.9/6.3.5. acetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), CO dehy 11), ferritin (EC 1.16.3.1), glutamate-1-semialdehyde 2.1- drogenase (EC 1.2.99.2), hydrogenase (EC 1.12.7.2). aminomutase (EC 5.4.3.8), glutamyl-tRNA reductase (EC pyruvate:formate lyase (EC 2.3.1.54), 2,3-butanediol dehy 1.2.1.70), glutamyl-tRNA synthetase (EC 6.1.1.17), drogenase (EC 1.1.1.4), primary:Seconday alcohol dehydro hydroxymethylbilane synthase (EC 2.5.1.61), nicotinate genase (EC 1.1.1.1), formate dehydrogenase (EC 1.2.1.2). nucleotide-dimethylbenzimidazole phosphoribosyltrans formyl-THF synthetase (EC 6.3.2.17), methylene-THF dehy ferase (EC 2.4.2.21), oxygen-independent coproporphyrino drogenase/formyl-THF cyclohydrolase (EC:6.3.4.3), meth gen III oxidase (EC 1.3.99.22), porphobilinogen synthase ylene-THF reductase (EC 1.1.1.58), CO dehydrogenase/ (EC 4.2.1.24), precorrin-2 dehydrogenase/sirohydrochlorin acetyl-CoA synthase (EC 2.3.1.169), CO dehydrogenase (EC ferrochelatase (EC 1.3.1.76/4.99.1.4), precorrin-2/cobalt 1.2.99.2), and hydrogenase (EC 1.12.7.2). factor-2 C20-methyltransferase (EC 2.1.1.130/2.1.1.151), 0061 An “enzyme co-factor” or simply a “co-factor” is a precorrin-3B synthase (EC 1.14.13.83), precorrin-3B C17 non-protein compound that binds to an enzyme to facilitate methyltransferase (EC 2.1.1.131), precorrin-4 C11-methyl the biological function of the enzyme and thus the catalysis of transferase (EC 2.1.1.133), precorrin-6X reductase (EC 1.3. a reaction. Non-limiting examples of co-factors include 1.54), precorrin-6YC5, 15-methyltransferase (EC 2.1.1.132), NAD+, NADP+, cobalamine, tetrahydrofolate and ferre precorrin-8W decarboxylase (EC 1.--...-), precorrin-8X meth doxin. “Nicotinamide adenine dinucleotide' (NADH) refers ylmutase (EC 5.4.1.2), sirohydrochlorin cobaltochelatase to either NAD+ (oxidized form), NADH--H+ (reduced form) (EC 4.99.1.3), threonine-phosphate decarboxylase (EC 4.1. or the the redox couple of both NAD+ and NADH--H+. 1.81), uroporphyrinogen decarboxylase (EC 4.1.1.37), “Nicotinamide adenine dinucleotide phosphate” (NADPH) uroporphyrinogen III methyltransferase/synthase (EC 2.1.1. refers to either NADP+ (oxidized form), NADPH-i-H+ (re 107/4.2.1.75). Without wishing to be bound by theory, it is duced form) or the redox couple of both NADP+ and believed that an increase in the availability of a co-factor is NADPH-i-H+. Increase in the overall availability of the co achieved through over-expression of enzymes or genes factor can increase the rate of a pathway reaction. Factors that involved in the biosynthesis pathway of said co-factor. As a may affect production of the co-factor include the expression result, reactions dependent on this co-factor are no longer of co-factor biosynthesis genes which may be altered to limiting. achieve increased availability of the co-factor. Other factors 0063. The invention also provides methods for the produc known to one of skill in the art may also be used to achieve tion of one or more products by fermentation of a substrate increased availability of the co-factor. Lack of availability of comprising CO. Preferably, the product is one or more of co-factors can have rate-limiting effects on pathway reac ethanol, butanol, isopropanol, isobutanol, higher alcohols, tions. Methods for the determination of availability of co butanediol. 2,3-butanediol. Succinate, isoprenoids, fatty factors are known in the art. acids, biopolymers, and mixtures thereof. 0062. The recombinant bacterium of the invention may 0064. In one embodiment, the substrate comprising CO is further express or be engineered to express or overexpress an a gaseous Substrate comprising CO. In one embodiment, the enzyme involved in the biosynthesis of a co-factor. In a par Substrate will typically contain a major proportion of CO. ticular embodiment, the co-factor comprises tetrahydro such as about 20% to about 100% CO by volume, from 20% folate. Enzymes that are involved in the biosynthesis of tet to 70% CO by volume, from 30% to 60% CO by volume, or rahydrofolate are detailed below. Accordingly, in a particular from 40% to 55% CO by volume. In particular embodiments, embodiment, the recombinant microorganism exhibits the substrate comprises about 25%, about 30%, about 35%, increased expression of GTP cyclohydrolase I (EC 3.5.4.16), about 40%, about 45%, about 50% CO, about 55% CO, or alkaline phosphatase (EC 3.1.3.1), dihydroneopterinaldolase about 60% CO by volume. (EC 4.1.2.25), 2-amino-4-hydroxy-6-hydroxymethyldihy 0065 While it is not necessary for the substrate to contain dropteridine diphosphokinase (EC 2.7.6.3), dihydropteroate any hydrogen, the presence of hydrogen should not be detri synthase (2.5.1.15), dihydropteroate synthase (EC 2.5.1.15), mental to product formation in accordance with methods of dihydrofolate synthase (EC 6.3.2.12), folylpolyglutamate the invention. In particular embodiments, the presence of synthase (6.3.2.17), dihydrofolate reductase (EC 1.5.1.3), hydrogen results in an improved overall efficiency of alcohol thymidylate synthase (EC 2.1.1.45), dihydromonapterin production. For example, in particular embodiments, the Sub reductase (EC 1.5.1.-). In a particular embodiment, the co strate may comprise an approx. 2:1, or 1:1, or 1:2 ratio of factor comprises cobalamine (B). Enzymes that are H:CO. In one embodiment the substrate comprises about involved in the biosynthesis of cobalamine are detailed below. 30% or less H by volume, 20% or less H. by volume, about Accordingly, in a particular embodiment, the recombinant 15% or less H by volume or about 10% or less H by volume. microorganism exhibits increased expression of 5-aminole In other embodiments, the Substrate stream comprises low Vulinate synthase (EC 2.3.1.37), 5-aminolevulinate:pyruvate concentrations of H, for example, less than 5%, less than 4%. aminotransferase (EC 2.6.1.43), adenosylcobinamide kinase/ less than 3%, less than 2%, or less than 1% H. In further adenosylcobinamide-phosphate guanylyltransferase (EC 2.7. embodiments, the Substrate stream is Substantially hydrogen 1.156/2.7.7.62), adenosylcobinamide-GDP ribazoletrans free. The Substrate may also contain some CO, for example, ferase (EC 2.7.8.26), adenosylcobinamide-phosphate about 1% to about 80% CO, by volume, or 1% to about 30% synthase (EC 6.3.1.10), adenosylcobyric acid synthase (EC CO by volume. In one embodiment the substrate comprises 6.3.5.10), alpha-ribazole phosphatase (EC 3.1.3.73), cob(I) less than or equal to about 20% CO by volume. In particular alaminadenosyltransferase (EC 2.5.1.17), cob(II)yrinic acid embodiments, the Substrate comprises less than or equal to a,c-diamide reductase (EC 1.16.8.1), cobalt-precorrin 5A about 15% CO by volume, less than or equal to about 10% US 2016/0160223 A1 Jun. 9, 2016

CO by volume, less than or equal to about 5% CO by 0070 The fermentation should desirably be carried out Volume, or Substantially no CO. under appropriate fermentation conditions for the production 0.066 Embodiments of the invention are described in of the fermentation product to occur. Reaction conditions that terms of delivering and fermenting a "gaseous Substrate con should be considered include pressure, temperature, gas flow taining CO. However, it should be appreciated that the gas rate, liquid flow rate, media pH, media redox potential, agi eous substrate may be provided in alternative forms. For tation rate (if using a continuous stirred tank reactor), inocu example, the gaseous Substrate containing CO may be pro lum level, maximum gas Substrate concentrations to ensure vided dissolved in a liquid. Essentially, a liquid is saturated that CO in the liquid phase does not become limiting, and with a carbon monoxide containing gas and then that liquid is maximum product concentrations to avoid product inhibition. added to the bioreactor. This may be achieved using standard (0071. In addition, it is often desirable to increase the CO methodology. By way of example, a microbubble dispersion concentration of a Substrate stream (or CO partial pressure in generator (Hensirisak, Appl Biochem Biotechnol, 101: 211 a gaseous Substrate) and thus increase the efficiency offer 227, 2002) could be used. By way of further example, the mentation reactions where CO is a Substrate. Operating at gaseous Substrate containing CO may be adsorbed onto a increased pressures allows a significant increase in the rate of Solid Support. Such alternative methods are encompassed by CO transfer from the gas phase to the liquid phase where it the term “substrate comprising CO' and the like. can be taken up by the micro-organism as a carbon Source for 0067. The gaseous substrate may be a CO-containing the production of fermentation. This in turn means that the waste gas obtained as a by-product of an industrial process or retention time (defined as the liquid volume in the bioreactor from Some other source. Such as from automobile exhaust divided by the input gas flow rate) can be reduced when fumes or biomass gasification. In certain embodiments, the bioreactors are maintained at elevated pressure rather than industrial process is selected from the group consisting of atmospheric pressure. The optimum reaction conditions will ferrous metal products manufacturing, such as a steel mill depend partly on the particular micro-organism of the inven manufacturing, non-ferrous products manufacturing, petro tion used. However, in general, it is preferred that the fermen leum refining processes, coal gasification, electric power pro tation be performed at pressure higher than ambient pressure. duction, carbon black production, ammonia production, Also, since a given CO conversion rate is in part a function of methanol production, and coke manufacturing. In these the Substrate retention time, and achieving a desired retention embodiments, the CO-containing gas may be captured from time in turn dictates the required volume of a bioreactor, the the industrial process before it is emitted into the atmosphere, use of pressurized systems can greatly reduce the Volume of using any convenient method. The CO may be a component of the bioreactor required, and consequently the capital cost of syngas (gas comprising carbon monoxide and hydrogen). The the fermentation equipment. According to examples given in CO produced from industrial processes is normally flared off U.S. Pat. No. 5,593,886, reactor volume can be reduced in to produce CO and therefore the invention has particular linear proportion to increases in reactor operating pressure, utility in reducing CO greenhouse gas emissions and pro i.e. bioreactors operated at 10 atmospheres of pressure need ducing a biofuel. Depending on the composition of the gas only be one tenth the volume of those operated at 1 atmo eous CO-containing Substrate, it may also be desirable to treat sphere of pressure. it to remove any undesired impurities, such as dust particles 0072 By way of example, the benefits of conducting a before introducing it to the fermentation. For example, the gas-to-ethanol fermentation at elevated pressures has been gaseous Substrate may be filtered or scrubbed using known described. For example, WO 2002/008438 describes gas-to methods. ethanol fermentations performed under pressures of 30 psig 0068 Typically, the fermentation is performed in a biore and 75 psig, giving ethanol productivities of 150 g/l/day and actor. The term “bioreactor” includes a fermentation device 369 g/l/day respectively. However, example fermentations consisting of one or more vessels and/or towers or piping performed using similar media and input gas compositions at arrangements, such as a continuous stirred tank reactor atmospheric pressure were found to produce between 10 and (CSTR), immobilized cell reactor (ICR), trickle bed reactor 20 times less ethanol per litre per day. (TBR), bubble column, gas lift fermenter, static mixer, or 0073. It is also desirable that the rate of introduction of the other vessel or other device Suitable for gas-liquid contact. In CO-containing gaseous Substrate is controlled to ensure that Some embodiments the bioreactor may comprise a first the concentration of CO in the liquid phase does not become growth reactor and a second fermentation reactor. As such, limiting, since products may be consumed by the culture when referring to the addition of substrate to the bioreactor or under CO-limited conditions. fermentation reaction it should be understood to include addi 0074 The composition of gas streams used to feed a fer tion to either or both of these reactors, where appropriate. As mentation reaction can have a significant impact on the effi used herein, the terms “fermenting.” “fermentation process.” ciency and/or cost of the reaction. For example, O may “fermentation reaction.” and the like encompass both the reduce the efficiency of an anaerobic fermentation process. growth phase and product biosynthesis phase of the fermen Processing of unwanted or unnecessary gases in stages of a tation process. fermentation process before or after fermentation can 0069. In certain embodiments a culture of a bacterium of increase the burden on Such stages. For example, where the the invention is maintained in an aqueous culture medium that gas stream is compressed before entering a bioreactor, unnec contains nutrients, vitamins, and/or minerals sufficient to per essary energy may be used to compress gases that are not mit growth of the microorganism. Preferably the aqueous needed in the fermentation. Accordingly, it may be desirable culture medium is a minimal anaerobic microbial growth to treat Substrate streams, particularly Substrate streams medium. Suitable media are known in the art and described derived from industrial sources, to remove unwanted compo for example in U.S. Pat. No. 5,173,429, U.S. Pat. No. 5,593, nents and increase the concentration of desirable compo 886, and WO 2002/008438. nentS. US 2016/0160223 A1 Jun. 9, 2016

EXAMPLES through a French press. Unbroken cells and cell debris were removed by centrifugation at 20,000xg and 4°C. for 30 min. 0075. The following examples further illustrate the inven The Supernatant was used for enzyme assays. Except where tion but, of course, should not be construed to limit its scope indicated, all assays were performed at 37° C. in 1.5-ml in any way. anaerobic cuvettes closed with a rubber stopper filled with 0.8 mlreaction mixture and 0.7 ml Nor H or CO at 1.2x10 Pa. Example 1 Enzymes were assayed as described below or by Huang, J 0076. This example describes the analysis offermentation Bacteriol, 194; 3689-3699, 2012. After the start of the reac pathways of carboxydotrophic bacteria Such as C. autoetha tion with enzyme, the reduction of NAD(P)" or NAD" was nogenium, C. liungdahli, or C. ragsdalei for bottlenecks in monitored spectrophotometrically at 340 nm (e=6.2 mM the production of ethanol and 2,3-butanediol. cm) or at 380 nm (e=1.2 mM cm), C. pasteurianum 0077. Oxidoreductase enzyme steps of the Wood ferredoxin reduction at 430 nm (eAa13.1 mM cm), Ljungdahl pathway and fermentation pathways to ethanol methyl viologen reduction at 578 nm (6–9.8 mM cm) and and 2,3-butanediol were assayed to determine their activity. benzyl viologen reduction at 578 nm (e=8.6 mM cm). Oxidoreductase reactions are particularly Suitable since they 0082 CO dehydrogenase was measured using an assay are coupled with one or more co-factors whose reduction or mixture that contained 100 mM Tris/HCl (pH 7.5), 2 mM oxidation can be measured. A synthetic redox dye such as DTT and about 30 uM ferredoxin and/or 1 mM NAD" or 1 methylviologen or benzylviologen can be used for this pur mM NADP". The gas phase was 100% CO. 0083 Hydroge pose as well. The enzymes in these pathways are involved in nase activity was measured using an assay mixture of 100 autotrophic growth including uptake and utilization of CO. mM Tris/HCl (pH 7.5) or 100 mM potassium phosphate, 2 CO, and H gases, as well as product formation. mMDTT and, 25uM ferredoxin and/or 1 mMNADP" and/or 0078. The enzymes assayed and their activities are 10 mM methyl viologen. The gas phase was 100% H. detailed in FIG. 1. All assays were performed using a syn I0083. Formate-hydrogen lyase activity for reduction of thetic redox dye as control, either methyl viologen (MV) or CO with H to formate was measured with an assay mixture benzyl viologen (BV). Co-factors ferredoxin (Fd), NADH, containing 100 mM potassium phosphate, 2 mM DTT, and 30 and NADPH or a combination thereof were then tested. mM (CKCO, (24,000 dpm/umol). The gas phase was Enzyme assays were performed using crude extracts from a 100% H. The serum bottles were continuously shaken at 200 fermentation growing autotrophically on CO and hydrogen. rpm to ensure equilibration of the gas phase with the liquid 007.9 Fermentations with C. autoethanogenium were car phase. After start of the reaction with enzyme, 100 ul liquid ried out in 1.5 L bioreactors at 37° C. using CO-containing samples were withdrawn every 1.5 min and added into a steel mill gas as a sole energy and carbon Source. The fermen 1.5-ml safe seal micro tube containing 100 ul of 150 mM tation media contained, per litre, MgCl, CaCl (0.5 mM), KCl acetic acid to stop the reaction by acidification. The 200 ul (2 mM), HPO (5 mM), Fe (100 uM), Ni, Zn(5uM), Mn, B, mixture was then incubated at 40°C. for 10 min with shaking W. Mo, and Se (2 uM). The media was transferred into a at 1,400 rpm in a Thermomixer to remove all ''CO, leaving bioreactor and autoclaved at 121° C. for 45 minutes. After behind the ''C-formate formed. Subsequently, 100 ul of the autoclaving, the media was Supplemented with thiamine, mixture was added to 5 ml of Quicksave A scintillation fluid pantothenate (0.05 mg) and biotin (0.02 mg) and reduced (Zinsser Analytic, Frankfurt, Germany) and analyzed for 'C with 3 mM cysteine-HC1. To achieve anaerobic conditions, radioactivity in a Beckman LS6500 liquid scintillation the reactor vessel was sparged with nitrogen through a 0.2 Lum counter (Fullerton, Calif.). filter. Prior to inoculation, the gas was switched to CO-con taining steel mill gas, feeding continuously to the reactor. The I0084. Formate dehydrogenase measurement was carried feed gas composition was 2% H, 42% CO, 20% CO, and out with an assay mixtures containing 100 mM Tris/HCl (pH 36% N. The pH of the culture was maintained between 5 and 7.5) or 100 mM potassium phosphate, 2 mM DTT, 20 mM 5.2. formate and, where indicated 25 uM ferredoxin, 1 mM 0080. At the time of harvesting the cells (biomass of 3.9 g, NADP", 1 mM NAD" and/or 10 mM methyl viologen. The cells/1 fermentation broth), the gas consumption was 5 moles gas phase was 100% N. COL' day' and 10 milimoles H. L.' day', with the fol I0085 Methylene-HF dehydrogenase was measured lowing metabolites produced: 14 g L' day' acetate and 19.5 using an assay mixture containing 100 mMMOPS/KOH (pH g L' day' ethanol. The pH of the culture was adjusted to pH 6.5), 50 mM 2-mercaptoethanol, 0.4 mMtetrahydrofolate, 10 6 with KCO and the reactor chilled in an ice-water bath. mM formaldehyde and 0.5 mM NADP" or 0.5 mM NAD". Approximately 1.2 L of culture was collected on ice. The The gas phase was 100% N. culture was divided between two 1-L centrifuge bottles (this I0086 Methylene-HF reductase was assayed under the and all Subsequent steps were carried out in an anaerobic following conditions. The assay mixtures contained 100 mM chamber to ensure anoxic conditions to avoid inactivation of Tris/HCl (pH 7.5), 20 mMascorbate, 10 uM FAD. 20 mM the enzymes) and cells were pelleted at 5000 rpm for 10 min. benzyl viologen and 1 mM methyl-H.F. Before start of the The Supernatant was decanted and residual liquid removed. reaction with enzyme, benzyl viologen was reduced to an Each pellet was resuspended in approximately 30 mL of 50 AA555 of 0.3 with sodium dithionite. mM K PO. pH 7.0 with 10 mM DTT. Resuspensions were transferred to pre weighed 50-mL-Falcon-tubes and cells I0087 Aldehyde:ferredoxin oxidoreductase was assayed repelleted at maximum speed (5000 g) for 15 min. The tubes using a mixture containing 100 mM Tris/HCl (pH 7.5), 2 mM were removed from the anaerobic chamber and immediately DTT, 1.1 mMacetaldehyde, and about 25uMferredoxin. The frozen on liquid N. before assaying. gas phase was 100% N. 0081 Cells were harvested from a continuous reactor I0088 CoA acetylating acetaldehyde dehydrogenase was under anoxic conditions. They were disrupted by three passes measured using a mixture contained 100 mM Tris/HCl (pH US 2016/0160223 A1 Jun. 9, 2016

7.5), 2 mM DTT, 1.1 mM acetaldehyde, 1 mM coenzyme A. Nhel (Fermentas), and the PFOR gene is ligated into and 1 mM NADP+ or 1 mMNAD+. The gas phase was 100% pMTL83155 with T4 DNA (Fermentas). The ligation N. mix is used to transform E. coli TOP10 (Invitrogen, LifeTech 0089 Alcohol and butanediol dehydrogenases were mea nologies) and colonies containing the desired plasmid are sured in an assay with 100 mM potassium phosphate (pH 6), identified by plasmid miniprep (Zymo Research) and restric 2 mM DTT, 1.1 mMacetaldehyde or acetoin respectively and tion digestion (Fermentas). The desired plasmid is methy 1 mMNADPH or 1 mMNADH. The gas phase was 100% N. lated and transformed in C. autoethanogenium. Successful 0090. Ferredoxin was purified from C. pasteurianum as transformants are identified by thiamphenicol resistance and described by Schönheit, FEBS Lett, 89: 219-222, 1978. PCR analysis with primers repHF and CatR which will yield 0091 All oxidoreductase reactions in the pathways to a 1584 product when the plasmid is present. ethanol and 2,3-butanediol of carboxydotrophic bacterium C. 0099 Transformants identified as containing the desired autoethanogenium were assayed and Successfully detected, plasmid are grown in serum bottles containing PETC-MES with the exception of the methylene-THF reductase which the media in the presence of mill gas, and their metabolite pro inventors believe requires an as yet unknown coupling site duction, measured by HPLC analysis, is compared to that of (Köpke, PNAS USA, 107: 13087-13092, 2010: Poehlein, a parental microorganism not harbouring the plasmid. The PLoS One, 7: e33439, 2012). Activity of this enzyme could pyruvate:ferredoxin oxidoreductase activity in the trans not previously be detected in other organisms. Results are formed strain is also measured in crude extracts to confirm provided in FIG. 1 and FIG. 2. This data was used to analyze that the observed bottleneck in the parental strain is allevi and determine bottlenecks in these pathways that would typi ated. Overexpression of pyruvate:ferredoxin oxidoreductase cally occur during a fermentation process. increases the overall activity within the cell, alleviating the bottleneck in the pathway, and leading to an increase in the Example 2 flux through pyruvate, and an increase in 2,3-butanediol pro 0092. This example demonstrates increasing the flux duction. through a fermentation pathway. 0093. The general methods described in Example 3 of Example 5 PCT/US2014/041188 may also be used to introduce pyru 0100. This example demonstrates an increase in flux from Vate:ferredoxin oxidoreductase, acetolactate synthase, and/or pyruvate to 2-hydroxy-2-methyl-3-ketobutyrate (acetolac acetolactate decarboxylase gene into the recombinant tate) via overexpression of native catabolic acetolactate Syn Clostridium microorganism of the invention. thase. 0101 The native catabolic acetolactate synthase gene Example 3 (alsS) of C. autoethanogenium is cloned into the Ndel and 0094. This example identifies the conversion of acetyl Nhel sites of pMTL83155 (WO 2013/185123) to generate an CoA to pyruvate by pyruvate:ferredoxin oxidoreductase as a overexpression plasmid, expressing alsS under the control of bottleneck in the production of 2,3-butanediol. the promoter region of the phosphotransacetylase-acetate 0095. As seen in FIG. 2, the bottleneck for 2,3-butanediol kinase . production is the reaction from acetyl CoA to pyruvate cata 0102 The overexpression plasmid can be similarly pro lyzed by pyruvate:ferredoxin oxidoreductase. While all other duced using a catabolic acetolactate synthase from another measured reactions showed at least an activity of 1.1 U/mg, microorganism, a native anabolic acetolactate synthase, oran this rate limiting reaction exhibited an enzyme activity of anabolic acetolactate synthase from another microorganism. only 0.11 U/mg (10%) in the presence of ferredoxin. This is 0103) The use of either a catabolic acetolactate synthase 90% less than all other reactions in the pathway. To go at least from another microorganism or an anabolic acetolactate Syn Some way towards overcoming this bottleneck and increase thase from another microorganism may have a higher affinity the product yield from the fermentation, an endogenous pyru toward pyruvate and faster reaction kinetics. An anabolic Vate:ferredoxin oxidoreductase enzyme may be overex acetolactate synthase from another microorganism may bean pressed or an exogenous pyruvate:ferredoxin oxidoreductase enzyme which is identified to be insensitive to feedback inhi enzyme may be introduced and expressed. bition. The small subunit of the anabolic acetolactate syn thase mutant which is insensitive to feedback inhibitions may Example 4 also be overexpressed. 0096. This example demonstrates increasing the flux 0104. The overexpression plasmid is introduced into C. through the 2,3-butanediol production pathway by removing autoethanogenium. This results in a C. autoethanogenium bottlenecks. strain adapted to increase flux from pyruvate to acetolactate. 0097. The reaction catalysing the conversion of acetyl Example 6 CoA to pyruvate has been identified in FIG. 2 to be the rate limiting step in 2,3-butanediol formation in C. autoethano 0105. This example describes a metabolic engineering genum, C. liungdahli, or C. ragsdalei. This can be overcome approach to overexpression of pyruvate:ferredoxin oxi by overexpressing the gene that encodes pyruvate:ferredoxin doreductase, acetolactate synthase, and/or acetolactate decar oxidoreductase in C. autoethanogenium. boxylase. 0098. The gene is codon-optimized to minimize issues 0106. In order to boost 2,3-butanediol production, the pool with expression and designed to reduce homology to the of pyruvate, a precursor molecule of 2,3-butanediol, was native gene to prevent undesired integration events. The gene increased. The first target was the PFOR gene which encodes is flanked by restriction enzyme cut sites, Xbal (3'-end) and the PFOR enzyme, which catalyzes the conversion of acetyl Nhel (5'-end) for subcloning into pMTL83155. The synthe CoA to pyruvate. In the C. autoethanogenium genome, there sized construct and pMTL83155 are digested with Xbaland are two copies of the PFOR gene. It is known that the PFOR US 2016/0160223 A1 Jun. 9, 2016 gene (CAETHG 0928) is constitutively expressed at a high presence of oxygen which could be advantageous in commer level while the other gene (CAETHG 3029) is only up-regu cial anaerobe fermentation (Pieulle, J Bacteriol, 179: 5684 lated at the end of the growth in a batch culture (Köpke, Appl 5692, 1997). The alsS gene isolated from Bacillus substilis Environ Microbiol, 77: 5467-5475, 2011). Thus, the highly and two heterologous alsD genes isolated from Leuconostoc expressed PFOR gene was chosen to be overexpressed. lactis and from Aeromonas hydrophila were also tested. The 0107 Acetolactate synthase, which links two pyruvate als.S gene isolated from Bacillus substilis was used to con molecules to form C-acetolactate, is proposed to exist in three struct the 2,3-butanediol pathway in a number of heterolo forms coded by three different genes in C. autoethanogenium gous hosts (Ng, Microb Cell Factories, 11: 68, 2012; Oliver, and in closely related microorganisms (Köpke, PNAS USA, PNAS, 110: 1249-1254, 2013). The alsD isolated from Aero 107: 13087-13092, 2010; Köpke, Appl Environ Microbiol, monas hydrophila was shown to have highest enzyme activity 77: 5467-5475, 2011): one catabolic acetolactate synthase among several other heterogolous alsD isolated from other and two forms of anabolic acetolactate synthase. The alsS microorganisms in a recent study (Oliver, PNAS, 110: 1249 gene (CAETHG 1740) is predicted to code for the catabolic 1254, 2013). These properties make these genes the ideal acetolactate synthase and to be involved in the formation of candidates for genetic manipulation experiments. 2,3-butanediol. The two genes ilvBN (CAETHG 0406) and ilvH (CAETHG 0124) are predicted to code for anabolic Example 7 acetolactate synthases which are likely to be involved in the 0112 This example describes cloning, conjugation, and formation of branched chain amino acids. characterization of strains overexpressing pyruvate:ferre 0108 C.-acetolactate is decarboxylated to acetoin via the doxin oxidoreductase, acetolactate synthase, and/or acetolac activity of acetolactate decarboxylase encoded by the alsD tate decarboxylase. gene (CAETHG 2932). In other microorganisms, the alsD 0113 C. autoethanogenium strain LZ1561 (DSM23693) gene transcript levels and the enzyme activity have been was used in this research. Two E. coli donor strains were used found to be regulated by the concentration of branched-chain as tools for genetic manipulation; the TOP 10 (Invitrogen) amino acids present in the cell. It is still unknown if the strain was used for plasmid cloning and the CA434 strain was branched-chain amino acids in C. autoethanogenium produce used for conjugation with C. autoethanogenium. any feedback inhibition of the alsD gene transcription or the 0114 A Clostridium-E. coli shuttle plasmid series activity of the corresponding enzyme. pMTL83159 (4600 base pairs) was chosen to overexpress 0109 The reduction step from acetoin to 2,3-butanediol PFOR, alsS, and alsD genes (Heap, J Microbiol Meth, 78: by the action of 2,3-butanediol dehydrogenase (EC 1.1.1.4) is 79-85, 2009). The plasmid was designed to contain a Gram not a rate limiting step. This has been demonstrated in batch positive replicon, a Gram negative replicon, a traJ gene, an and continuous cultures of C. autoethanogenium by the addi antibiotic resistant gene, the multiple cloning sites located tion of acetointo the fermentation media. In batch cultures, up within the lacz alpha coding sequence, and the ferredoxin to 40 g/L of acetoin was added and quantitatively converted to gene promoter (P.). The Gram-positive replicon (repH 2,3-butanediol after 24 hours of incubation. In fact, the puta gene) originated from the C. butyllicum pCB102 plasmid. To tive 2,3-butanediol dehydrogenase gene was found to be clone the plasmid in E. coli the Gram-negative replicon expressed constitutively during both growth and Stationary ColE1 was chosen due to the high copy number of plasmids it phase in a batch culture (Köpke, Appl Environ Microbiol, 77: produces. A traJortransfer geneallows the genetic material to 5467-5475, 2011). Furthermore, it has been shown that C. be transferred between donor and recipient cell. The catP autoethanogenium contains a strictly NADPH-dependent pri gene encoded for the chloramphenicol/thiamphenicol resis mary-secondary alcohol dehydrogenase which also reduces tance was the selection marker. acetoin and other ketones to 2,3-butanediol and other second 0115 The three genes. PFOR, alsS, and alsD, were syn ary alcohols (Köpke, Catalyst Rev. 27: 7-12, 2014). thesized by a DNA synthesizing company (GeneArt). To 0110. The three native genes, PFOR, alsS, and alsD, were clone them into the pMTL83159 plasmid, a pair of restriction overexpressed individually and in combination of alsS-alsD enzyme recognition sequences and a ribosomal and in combination of all three genes. To introduce the genes sequence were added to each gene sequence and synthesized into C. autoethanogenium, the genes were cloned into a together with the gene. The original plasmids carrying the recombinant plasmid that carried an antibiotic resistant gene gene were first transformed into the E. coli TOP 10 strain. A as a selection marker. For this reason, the control strain for ZYPPYTM plasmid miniprep kit (Zymo Research) was used this set of experiments carried the plasmid with the antibiotic to extract the plasmids. To transfer the targeted gene from its resistance gene but without any active 2,3-butanediol gene original plasmid to a pMTL83159 plasmid, both plasmids insertion, so it could be exposed to antibiotic stress and com were cut with the same pair of restriction enzymes. The pared to the performance of the overexpression Strains. An digested DNA was separated by gel electrophoresis on a 0.6% important aspect of overexpression is the promoter choice to agarose gel which ran at 75 volts for one hour. After the regulate the expression of inserted genes. In this research, the plasmidpMTL83159 (vector) and the DNA sequence (insert) ferredoxin gene promoter (P.) was chosen as it is known to of each gene were recovered from the gel, they were ligated be one of the strongest promoters in Clostridia. Furthermore, together by using T4 DNA ligase (Invitrogen). The ligated to avoid homologous recombination between the native gene plasmids were then transformed into the E. coli Top 10 cul in the genome and the gene present in the plasmid, the DNA ture. To ensure Sure that the extracted plasmids contained the sequence of the added genes was altered according to a pro insert, 1 uL of the plasmid was digested with appropriate prietary optimizing process (GeneOptimizer) of the DNA restriction enzymes and the digested plasmid was then sepa synthesizing company (GeneArt). rated by gel electrophoresis on a 0.8% agarose gel. 0111 Four heterologous genes were also targeted. A 0116. The plasmids were transformed into E. coli CA434 PFOR gene isolated from Desulfo vibrio africanus has been donor cells and then conjugated with C. autoethanogenium. shown to produce a PFOR enzyme that is highly stable in the To confirm the presence of the targeted plasmid in the C. US 2016/0160223 A1 Jun. 9, 2016 autoethanogenium transformants, PCR was performed using alsD genealone. A positive additional effect of the alsS might samples taken from the cultures in serum bottles. be feasible. Overexpression of the PFOR gene appeared to 0117 The initial characterization of all the gene overex have contributed to a higher 2,3-butanediol production during pression and gene disruption strains were first performed in 1 the stationary phase where no more growth was observed. L-Schott bottles to screen which strain had produced the Because of all of these positive results and the fact that no highest concentration of 2,3-butanediol. These strains were detrimental effect had been observed in these strains, the then further tested in CSTR continuous cultures, which overexpression strain carrying all three gene was further allows for accurate control of fermentation parameters such tested in the continuous culture in CSTR. that metabolite and biomass selectivities can be calculated. 0.122 To explore the full potential of the microbe using a 0118. The goals of the overexpression experiments were CO-containing gaseous Substrate, the overexpression strain to overexpress three native genes in the 2,3-butanediol path carrying all three native genes and the plasmid control strain way individually and in combinations of two and three genes. were further characterized in CSTR-based continuous cul tures. The media pH was controlled during the entire fermen tation by adding a base (5 MNHOH-solution) to compensate Schott bottle for the acid production and replenish media nitrogen levels. Overexpression strain Strain availability data availability The Substrate was continuously supplied by sparging a CO Plasmid control containing gas mix through the stirred fermentation broth. Native PFOR The gas composition of the fresh incoming gas and the used Native AISS Native AlsD outflowing gas was monitored hourly by gas chromatogra Native Alss-AlsD phy. Based on the differences in gas composition between the Native PFOR-AlSS-AlsD inflowing and outflowing gases, the flow rate of the incoming gas, and the liquid Volume of the fermentation; the gas utili 0119. In Schott bottle experiments, the OD, metabolite Zation and rate of product synthesis at the time of sampling concentrations in the media, pH of the media and headspace were calculated. The values are expressed in mol/L/d. pressure were monitored over the course of eight days by (0123. The OD and metabolite concentrations were mea analyzing daily samples. At that point no further growth or sured three times a day and the dilution rate and the specific significant metabolic activity was observed, the pH of all the growth rate were measured and calculated daily to determine cultures had dropped to between 3.8 and 4, and no further gas the productivity of each metabolite. The product selectivity of consumption was measured. A graphic representation of the each metabolite was calculated using the CO consumption, growth and metabolite profiles versus time of the five strains CO production, and the metabolite productivity. CO uptake are shown in FIG. 5. All five strains with Schott bottle data rates of 4 mol/L/d and 8 mol/L/d were established to deter availability quickly grew during the first two days of incuba mine whether the product selectivity is dependent on the tion. Thereafter, the growth rate reduced significantly and volumetric productivity. At a CO uptake of 4 mol/L/d, the then ceased on day 4. The maximum optical density values dilution rate of the system was maintained at 1 d' and the were approximately 0.75. Similarly to the biomass, the acetic specific growth rate at 0.5 d'. At higher CO uptake of 8 acid concentrations increased steeply during the first two mol/L/d and correspondingly higher metabolite production days in all the cultures. Between day 3 and day 4, the plasmid rates the dilution rate of the culture was increased to 1.7 d' to control and the alss overexpression strains appeared to have lower the metabolite concentrations to a similar range as in stopped all metabolic activities, as no further changes in the 4 mol/L/d experiments. The specific growth rate was also metabolite concentrations were observed. The other three increased to 0.75 d'. strains showed activity until end of the experiments. 0.124. The metabolite and gas profile of the combined 0120 Conversion of acetate to ethanol (AOR activity) PFOR, alsS, and alsDoverexpression strain and the plasmid could still be observed in three strains after the growth slowed control strain was monitored at a 4 mol/L/d CO uptake over down. The most notable drop in acetate was observed in the the course of 20 days (FIG. 6). Although the preparation of the alsDoverexpression strain, and as a result, the highest ethanol inoculum of each strain went well through several rounds of concentration of around 7 g/L was reached by this strain. Two serum bottle sub-culturing in regular intervals, the CSTR strains, the alsD and the combined alsS-alsD overexpression cultures exhibited an unusual, almost identical long lag phase strains, produced higher amounts of 2,3-butanediol than the of five days. At around day 5.5, both CSTR cultures started other Strains, producing most of it during the active growth normal exponential growth (within hours of each other). phase. Thereafter, during the stationary phase from day 4 they 0.125 Despite the long lag phase in both cultures and the continued producing 2,3-butanediol at slower rates until the fluctuation in the growth pattern of the overexpression strain end of the experiment on day 8. The PFOR overexpression between days 8 and 10, both cultures were maintained at a strain was the other strain that produced 2,3-butanediol to a stable gas uptake of 4 mol/L/d for 10 days. With a dilution rate higher concentration than the plasmid control strain. In con of 1 d' and a specific growth rate of 0.5 d, it takes about six trast to the first two strains, the PFOR overexpression strain days to replace 95% of the bacterial load in the fermenters. started producing most of the 2,3-butanediol during the onset With the constant gas uptake measured during this period, the of the stationary phase. The production rate was similar to the latest values were used for analysis. The final results showed rates of the other two strains during the stationary phase. that the overexpression strain consistently produced higher 0121 Overexpression of the alsS gene alone did not 2,3-butanediol levels compared to the plasmid control strain. appear to increase the amount of 2,3-butanediol. These results 0.126 The metabolite and the gas profiles of the overex Suggest that the 2,3-butanediol increase observed is primarily pression culture was monitored at an 8 mol/L/d CO uptake associated with the overexpression of the alsD gene. Overex over the course of 11 days (FIG. 7). The culture was inocu pressing both alsS and alsD genes resulted in a slightly higher lated from the 4 mol/L/d CO uptake culture. No lag phase was 2,3-butanediol concentration than just overexpressing the observed in this culture, and an appropriate specific CO Sup US 2016/0160223 A1 Jun. 9, 2016 ply and dilution rate was applied during exponential growth expression in C. autoethanogenium (GeneArt) and cloned into to keep the culture under optimal growing conditions. There pMTL83159 as described above. The resulting plasmids, and fore a stable production of acetic acid was reached after day pMTL83159 as a control, were transformed into C. autoet three of incubation while other metabolites continued to hanogenium strain LZ1561 as described above. accumulate. The target of CO uptake was doubled from 4 I0132 Strains were grown in 1-L Schott bottles containing mol/L/d to 8 mol/L/d. 40 mL of PETC-MES medium with no yeast extract and the 0127. To avoid product inhibition, the overall dilution rate headspace was replaced with 1.5 bar(gauge) synthetic mill of the culture was increased from 1 d' to 1.7 d' and the gas (50% CO, 29% N, 18% CO, and 3% H) as the carbon specific growth rate was increased proportionally. Metabolite and energy source. To maintain the plasmid, 15 mg L' thia concentrations slowly increased and reached a stable produc mphenicol was added. Biomass and metabolite concentra tion rate after seven days. Both the hydrogen uptake and the tions were recorded through the growth of the cultures. CO uptake were maintained in a stable manner for six days, 0.133 Expression of either exogenous acetolactate decar indicating that the fermentation of the overexpression strain boxylase led to an increase of 2,3-butanediol production dur has the potential to be operated stably for extended time ing growth on synthetic mill gas as compared to the strain periods. harboring the empty plasmid as a control. Expression of alsD from A. hydrophila and L. lactis led to production of 2.3+0.08 0128. Among the liquid products, ethanol was produced at and 1.6+0.16 g L 2,3-butanediol, respectively, compared to the highest rate followed by acetate. The specific CO supply a production of 0.3+0.12g L' by the empty-plasmid-control strategy was aimed to maintain a certain ratio of acetate to strain (FIG. 8). ethanol that allows the fermentation to be operated stably for 0.134 All references, including publications, patent appli extended periods of time. The LZ1561 strain and the plasmid cations, and patents, cited herein are hereby incorporated by control exhibited similar 2,3-butanediol and biomass profiles. reference to the same extent as if each reference were indi The 2,3-butanediol to biomass production rate ratios were vidually and specifically indicated to be incorporated by ref between 1.26 to 1.47 in these cultures. However, in the over erence and were set forth in its entirety herein. The reference expression strain, this ratio was 2.45 at 4 mol/L/d CO uptake to any prior art in this specification is not, and should not be and 2.24 at the 8 mol/L/d CO uptake culture. taken as, an acknowledgement that that prior art forms part of the common general knowledge in the field of endeavour in Over- Over any country. Strain LZ1561 Control expression LZ1561 expression 0.135 The use of the terms “a” and “an and “the and similar referents in the context of describing the invention Gas uptake rate (mol/Ltd) (especially in the context of the following claims) are to be CO consumption 4 4 4 8 8 construed to cover both the singular and the plural, unless CO2 production 2.45 2.53 2.43 4.53 4.34 otherwise indicated herein or clearly contradicted by context. Product Production rate (10 mol/L/d) The terms “comprising.” “having,” “including,” and “con 2,3-Butanediol 59 57 81 122 168 taining are to be construed as open-ended terms (i.e., mean Biomass 37 44 33 83 75 ing “including, but not limited to) unless otherwise noted. Ethanol 447 450 367 798 823 Recitation of ranges of values herein are merely intended to Acetic acid 128 133 125 258 214 serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indi 0129. The product selectivity for 2,3-butanediol, biomass, cated herein, and each separate value is incorporated into the ethanol and acetate for the overexpression, control and specification as if it were individually recited herein. All LZ1561 Strains was measured at a gas uptake of 4 mol/L/d methods described herein can be performed in any suitable and 8 mol/L/d. With the optimized fermentation parameters, order unless otherwise indicated herein or otherwise clearly more than 50% of the carbon was directed to ethanol forma contradicted by context. The use of any and all examples, or tion. The data shows that 2,3-butanediol selectivity of the exemplary language (e.g., “Such as”) provided herein, is overexpression strain increased from an average of 15% in the intended merely to better illuminate the invention and does LZ1561 and the plasmid control cultures to 22.5%. The not pose a limitation on the scope of the invention unless elevated 2,3-butanediol selectivity of the overexpression otherwise claimed. No language in the specification should be strain appeared to be maintained at different CO uptake rates. construed as indicating any non-claimed element as essential The increase in 2,3-butanediol selectivity is contributed to the to the practice of the invention. decrease of ethanol selectivity at 4 mol/L/d or the decrease in 0.136 Preferred embodiments of this invention are the acetate selectivity at 8 mol/L/d. The exact contribution of described herein, including the best mode knownto the inven ethanol and acetate cannot be separated, because their selec tors for carrying out the invention. Variations of those pre tivity can be influenced easily by the specific gas Supplies, ferred embodiments may become apparent to those of ordi which in turn are easily influenced by small differences nary skill in the art upon reading the foregoing description. between run parameters. For instance, pH, impeller position, The inventors expect skilled artisans to employ such varia probe location among others, can all affect these results. tions as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described Example 8 herein. Accordingly, this invention includes all modifications and equivalents of the Subject matter recited in the claims 0130. This example demonstrates expression of exog appended hereto as permitted by applicable law. Moreover, enous acetolactate decarboxylase to increase flux toward 2.3- any combination of the above-described elements in all pos butanediol. sible variations thereof is encompassed by the invention 0131 Acetolactate decarboxylase genes from A. hydro unless otherwise indicated herein or otherwise clearly con phila and L. lactis were synthesized with codons selected for tradicted by context. US 2016/0160223 A1 Jun. 9, 2016 14

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 28

<210s, SEQ ID NO 1 &211s LENGTH: 351.3 &212s. TYPE: DNA <213> ORGANISM: Clostridium autoethanogenum LZ1561

<4 OOs, SEQUENCE: 1 atgcgtaaaa tdaaaact at gigatggaaat actgctgctg. cittatatttic ctatogcattt 6 O actgatgtag cagctattta t ccaataact c catcat cac caatggcaga acatgtagat 12 O gaatgggtag cta agggaaa gaaaaatatt tttggacaac alagtaaaagt tatggagatg 18O

Caat cagagg Ctggagcatc aggagc.cgta catggttcac tacaa.gcagg agcattgact 24 O agtacatata citgcatct ca aggct tatta cittatgatac cta acatgta taagattgct 3OO ggtgaattat taccaggagt attic catgta t cagotagag cagtagctgc aaatt cactt 360 aacatatttg gtgat cacca agatgttatg gcaacaagac aaactggatt togctittattt 42O gcagaaagtt cagtacaa.ca ggittatggat ctitt cagcag tagcc cattt at cagcaata 48O aaaggaagag titc catttgt aaactitctitt gatggattica gaactitctica tdaaattcaa 54 O aaaatcgaat tattagagta tdaagaatta gcaaaattag ttgat cagaa agctittgaat 6OO gattittagaa gaagagctitt aaatccagat catccagtaa citcgtgg tac agctcaaaat 660 cCtgat at at act tcc agga aagagaagtt t caaatacgt attacgaagic act tccagag 72 O at agttgaag gatatatgca agaaatgact aaact tacag gaagagaata t catctatt c 78O aattactatg gagcaaaaga tigcagagaga attataatag ctatgggttctgtctgtgaa 84 O actgtagaag aagtaattga t tact taacc acaaaaggtgaaaaagttgg attact taca 9 OO gttcatttat atagac catt citcaataaaa cactittatga aatacatacc aaagactgtt 96.O aagaaaattig cagtcc ttga tagaacaaaa gaaccaggat caattggaga acct ctititat O2O ttagatgtta agaatgctitt Ctatggacaa gaagtacaac Cagttatagt tdgtggalaga O8O tatggactitg gttcaaaaga tigt attacca toacatatto taccagtatt tdaaaactta 14 O aaat cagata aacctaagga tagatttaca ttalagtatag ttgatgatgt tacaaatact 2OO t cattacctg taggagaaga tataaataca acaccagaag gaact acagc titgitaagttc 26 O tggggactag gat Cagatgg tactgttgga gcaaacaaga gtgctataala gat cattgga 32O gaccatacag atatgtatgc ticaaggatac tittgcatatg attcaaagaa atc.cggtggit 38O ataa.cagttt ct catttaag atttggtaaa tolaccaataa aat caccata t cittatagat 44 O aaggctgatt ttgttgcagt toatalaccaa tottatgttc ataagtatga tigtacttgca SOO ggacttaaaa aaggcgg tala Cttct tatta aatacagttt ggact caaga agaattggaa 560 aaagagttac cagctt citat gaagaaatat at agcaaaca atgatataaa attctataca 62O ttaaatgctg. ittaaaatagc ticaagaaatt ggacttggtg gaagaataaa tatgatatgt 68O caat cago at t ctittaagat togcaaatat c attccaatag atgatgctgt taagtactta 74 O aaagaa.gcag ttgtaact tc titatggtaag aagggacaaa aagttgtaga tatgaataac 8OO gctgctatag acaagggcgt aaatgcagta gttaaaatag atgttccago tt catggaaa 86 O gatgcagaag atgaag cagc agctacaaag gaact tccta aatttataga aaaaatagitt 92 O aatcct atga at agaCaaga aggagacaaa Ctt coagtaa gtgcatttgt aggaatggaa 98 O US 2016/0160223 A1 Jun. 9, 2016 15

- Continued gatggtacat tcc cagcagg aactgcagct tatgaaaaga gaggaat agc tataaatgtt 2O4. O ccagaatggc aagtag acaa atgtatacaa totaaccaat gttcatttgt atgtccacat 21OO gcagotataa gaccagttct tacaactgaa gaagaattag ctaaag.cacc ticaaggattit 216 O gaagctaaag atgcaaatgg agcaaaagga cittaaattta caatggctat titcaccactt 222 O gattgttcag gatgtggaala Ctgttgaagat gtatgtc.cag caaaagaaaa ggct Cttgtt 228O atgaagc.cag tagatactica gctgtcaaag acagaagctt gggattatgc tigttaatgct 234 O gtagct cata aggata accc aatgaaggac aaatacagtg taaaa.gcaa.g. t cagttctgaa 24 OO

Caac Cattac ttgagttctic aggagcttgt gcaggatgcg gagaaact cc titatgttaaa 246 O cittgta actic aattgtttgg tdatagaatg atgatagcaa atgctacagg atgttcatca 252O atttggggag cat cagcacc agcaacticca tacacaacta act at agagg acatggit coa 2580 t cittgggcta act cattatt tdaagacaat gctgaatatg gattaggitat gttcc ttgga 264 O gttaalacaga caa.gagaaag actticaggat aaaattgaag aagctittaaa ggg tagttta 27 OO agtgcagaac ttaaagctgc titttgaagac tattaaaa actittgctga aggtgaagga 276 O acaa.gaga aa gagctgataa aataa.cagct ttacttgaaa aagaaaaggg alagcaatgat 282O ttattaaatg at atttatga aaacagagac titcc tagtaa agagat.ccca citggataatt 288O ggtggagacg gttggggota tatattgga tatggtggag tagat catgt tittagct tca 294 O aatgaagatg taaatatt ct td tacttgat acagaagitat attcaaatac aggtggacaa 3 OOO tottcaaaat caactic caac agctgctgta gctaaatttg citgcaagtgg taagaag act 3 O 6 O aagaagaaag at Cttggaat gatggctatg agittatggat atgtt tatgt agcticagatt 312 O t caatgggtg Ctgataagaa t caggcatta aaggcaattic atgaagcaga agctt at Cat 318O ggaccatcac ttataatago titatgcticca totat caatc atggcttaag agttggaatg 324 O ggtaagagcc agagagaa.gc taagagagct gttgattgttg gat attgggc actitt acaga 33 OO tacaatccag aattaaaaga agaaggaaag aaatcattta gcttggattic aaaagaacca 3360 actacagatt toaaggaatt cittaatggga gaagtaagat act citt cact togctaaacaa 342O titcc cagat.c aggcagatgc attatttgaa aagactaaga aagatgctict tcaaagaatt 3480 gCaggatata aaaagcttga taatgaacag taa 35.13

<210s, SEQ ID NO 2 &211s LENGTH: 117O 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum LZ1561

<4 OOs, SEQUENCE: 2 Met Arg Llys Met Lys Thr Met Asp Gly Asn. Thir Ala Ala Ala Tyr Ile 1. 5 1O 15

Ser Tyr Ala Phe Thr Asp Val Ala Ala Ile Tyr Pro Ile Thr Pro Ser 2O 25 3O Ser Pro Met Ala Glu. His Val Asp Glu Trp Val Ala Lys Gly Lys Llys 35 4 O 45

Asn Ile Phe Gly Glin Glin Val Llys Val Met Glu Met Glin Ser Glu Ala SO 55 6 O

Gly Ala Ser Gly Ala Wal His Gly Ser Lieu. Glin Ala Gly Ala Lieu. Thir 65 70 7s 8O

Ser Thr Tyr Thr Ala Ser Glin Gly Lieu. Leu Lleu Met Ile Pro Asn Met US 2016/0160223 A1 Jun. 9, 2016 16

- Continued

85 90 95 Tyr Lys Ile Ala Gly Glu Lieu. Lieu Pro Gly Val Phe His Val Ser Ala 1OO 105 11 O Arg Ala Val Ala Ala Asn. Ser Lieu. Asn. Ile Phe Gly Asp His Glin Asp 115 12 O 125 Val Met Ala Thr Arg Glin Thr Gly Phe Ala Leu Phe Ala Glu Ser Ser 13 O 135 14 O Val Glin Glin Val Met Asp Lieu. Ser Ala Val Ala His Lieu. Ser Ala Ile 145 150 155 160 Lys Gly Arg Val Pro Phe Val Asin Phe Phe Asp Gly Phe Arg Thr Ser 1.65 17O 17s His Glu Ile Glin Lys Ile Glu Lieu. Lieu. Glu Tyr Glu Glu Lieu Ala Lys 18O 185 19 O Lieu Val Asp Gln Lys Ala Lieu. Asn Asp Phe Arg Arg Arg Ala Lieu. Asn 195 2OO 2O5 Pro Asp His Pro Val Thr Arg Gly Thr Ala Glin Asn Pro Asp Ile Tyr 21 O 215 22O Phe Glin Glu Arg Glu Val Ser Asn Thr Tyr Tyr Glu Ala Leu Pro Glu 225 23 O 235 24 O Ile Val Glu Gly Tyr Met Glin Glu Met Thr Lys Lieu. Thr Gly Arg Glu 245 250 255 Tyr His Lieu. Phe Asn Tyr Tyr Gly Ala Lys Asp Ala Glu Arg Ile Ile 26 O 265 27 O Ile Ala Met Gly Ser Val Cys Glu Thr Val Glu Glu Val Ile Asp Tyr 27s 28O 285 Lieu. Thir Thr Lys Gly Glu Lys Val Gly Lieu. Lieu. Thr Val His Lieu. Tyr 29 O 295 3 OO Arg Pro Phe Ser Ile Llys His Phe Met Lys Tyr Ile Pro Llys Thr Val 3. OS 310 315 32O Llys Lys Ile Ala Val Lieu. Asp Arg Thr Lys Glu Pro Gly Ser Ile Gly 3.25 330 335 Glu Pro Lieu. Tyr Lieu. Asp Wall Lys Asn Ala Phe Tyr Gly Glin Glu Val 34 O 345 35. O Glin Pro Val Ile Val Gly Gly Arg Tyr Gly Lieu. Gly Ser Lys Asp Wall 355 360 365 Lieu Pro Ser His Ile Lieu Pro Val Phe Glu Asn Lieu Lys Ser Asp Llys 37 O 375 38O Pro Lys Asp Arg Phe Thr Lieu. Ser Ile Val Asp Asp Val Thir Asn Thr 385 390 395 4 OO Ser Leu Pro Val Gly Glu Asp Ile Asn. Thir Thr Pro Glu Gly. Thir Thr 4 OS 41O 415 Ala Cys Llys Phe Trp Gly Lieu. Gly Ser Asp Gly Thr Val Gly Ala Asn 42O 425 43 O Llys Ser Ala Ile Lys Ile Ile Gly Asp His Thr Asp Met Tyr Ala Glin 435 44 O 445

Gly Tyr Phe Ala Tyr Asp Ser Lys Llys Ser Gly Gly Ile Thr Val Ser 450 45.5 460 His Lieu. Arg Phe Gly Llys Ser Pro Ile Llys Ser Pro Tyr Lieu. Ile Asp 465 470 47s 48O Lys Ala Asp Phe Val Ala Wal His Asn Glin Ser Tyr Val His Llys Tyr 485 490 495 US 2016/0160223 A1 Jun. 9, 2016 17

- Continued

Asp Val Lieu Ala Gly Lieu Lys Lys Gly Gly Asn. Phe Lieu. Lieu. Asn Thr SOO 505 51O Val Trp Thr Glin Glu Glu Lieu. Glu Lys Glu Lieu Pro Ala Ser Met Lys 515 52O 525 Llys Tyr Ile Ala Asn. Asn Asp Ile Llys Phe Tyr Thr Lieu. Asn Ala Val 53 O 535 54 O Lys Ile Ala Glin Glu Ile Gly Lieu. Gly Gly Arg Ile Asn Met Ile Cys 5.45 550 555 560 Glin Ser Ala Phe Phe Lys Ile Ala Asn. Ile Ile Pro Ile Asp Asp Ala 565 st O sts Val Llys Tyr Lieu Lys Glu Ala Val Val Thir Ser Tyr Gly Lys Lys Gly 58O 585 59 O Glin Llys Val Val Asp Met Asn. Asn Ala Ala Ile Asp Llys Gly Val Asn 595 6OO 605 Ala Val Val Lys Ile Asp Val Pro Ala Ser Trp Lys Asp Ala Glu Asp 610 615 62O Glu Ala Ala Ala Thr Lys Glu Lieu Pro Llys Phe Ile Glu Lys Ile Val 625 630 635 64 O Asn Pro Met Asn Arg Glin Glu Gly Asp Llys Lieu Pro Val Ser Ala Phe 645 650 655 Val Gly Met Glu Asp Gly Thr Phe Pro Ala Gly Thr Ala Ala Tyr Glu 660 665 67 O Lys Arg Gly Ile Ala Ile Asn Val Pro Glu Trp Glin Val Asp Llys Cys 675 68O 685 Ile Glin Cys Asn Glin Cys Ser Phe Val Cys Pro His Ala Ala Ile Arg 69 O. 695 7 OO Pro Val Lieu. Thir Thr Glu Glu Glu Lieu Ala Lys Ala Pro Glin Gly Phe 7 Os 71O 71s 72O Glu Ala Lys Asp Ala Asn Gly Ala Lys Gly Lieu Lys Phe Thr Met Ala 72 73 O 73 Ile Ser Pro Lieu. Asp Cys Ser Gly Cys Gly Asn. Cys Glu Asp Val Cys 740 74. 7 O Pro Ala Lys Glu Lys Ala Lieu Val Met Llys Pro Val Asp Thr Glin Lieu. 7ss 760 765 Ser Lys Thr Glu Ala Trp Asp Tyr Ala Val Asn Ala Val Ala His Lys 770 775 78O Asp ASn Pro Met Lys Asp Llys Tyr Ser Val Lys Ala Ser Glin Phe Glu 78s 79 O 79. 8OO Glin Pro Lieu. Lieu. Glu Phe Ser Gly Ala Cys Ala Gly Cys Gly Glu Thir 805 810 815 Pro Tyr Val Lys Lieu Val Thr Gln Leu Phe Gly Asp Arg Met Met Ile 82O 825 83 O

Ala Asn Ala Thr Gly Cys Ser Ser Ile Trp Gly Ala Ser Ala Pro Ala 835 84 O 845 Thr Pro Tyr Thr Thr Asn Tyr Arg Gly His Gly Pro Ser Trp Ala Asn 850 855 860

Ser Lieu. Phe Glu Asp Asn Ala Glu Tyr Gly Lieu. Gly Met Phe Lieu. Gly 865 87O 87s 88O

Val Lys Glin Thr Arg Glu Arg Lieu. Glin Asp Llys Ile Glu Glu Ala Lieu 885 890 895 US 2016/0160223 A1 Jun. 9, 2016 18

- Continued Lys Gly Ser Lieu. Ser Ala Glu Lieu Lys Ala Ala Phe Glu Asp Trp Ile 9 OO 905 91 O Lys Asn. Phe Ala Glu Gly Glu Gly Thr Arg Glu Arg Ala Asp Llys Ile 915 92 O 925 Thir Ala Lieu. Lieu. Glu Lys Glu Lys Gly Ser Asn Asp Lieu. Lieu. Asn Asp 93 O 935 94 O Ile Tyr Glu Asn Arg Asp Phe Lieu Val Lys Arg Ser His Trp Ile Ile 945 950 955 96.O Gly Gly Asp Gly Trp Gly Tyr Asp Ile Gly Tyr Gly Gly Val Asp His 965 97O 97. Val Lieu Ala Ser Asn. Glu Asp Val Asn. Ile Lieu Val Lieu. Asp Thr Glu 98O 985 99 O Val Tyr Ser Asn Thr Gly Gly Glin Ser Ser Lys Ser Thr Pro Thr Ala 995 1OOO 1005 Ala Val Ala Llys Phe Ala Ala Ser Gly Lys Llys Thr Lys Llys Llys O1O O15 O2O Asp Leu Gly Met Met Ala Met Ser Tyr Gly Tyr Val Tyr Val Ala O25 O3 O O35 Glin Ile Ser Met Gly Ala Asp Lys Asn Glin Ala Lieu Lys Ala Ile O4 O O45 OSO His Glu Ala Glu Ala Tyr His Gly Pro Ser Lieu. Ile Ile Ala Tyr O55 O6 O O65 Ala Pro Cys Ile Asn His Gly Lieu. Arg Val Gly Met Gly Llys Ser Of O O7 O8O Glin Arg Glu Ala Lys Arg Ala Val Asp Cys Gly Tyr Trp Ala Lieu. O85 O9 O O95 Tyr Arg Tyr Asn Pro Glu Lieu Lys Glu Glu Gly Llys Llys Ser Phe

Ser Lieu. Asp Ser Lys Glu Pro Thr Thr Asp Phe Lys Glu Phe Lieu.

Met Gly Glu Val Arg Tyr Ser Ser Lieu Ala Lys Glin Phe Pro Asp

Glin Ala Asp Ala Lieu. Phe Glu Lys Thir Lys Lys Asp Ala Lieu. Glin

Arg Ile Ala Gly Tyr Lys Llys Lieu. Asp Asn. Glu Glin 16 O 65 17 O

<210s, SEQ ID NO 3 &211s LENGTH: 3538 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized Clostridium autoethanogenum LZ1561 pyruvate : ferredoxin oxidoreductase

<4 OOs, SEQUENCE: 3 tctagaagga ggtaactaaa tagaaaaat gaaaact atg gatggaaata Ctgcagcagc 6 O atatataagt tatgcattta citgatgtagc agcaatatat cctataactic ctagtag to c 12 O tatggcagaa catgtagatgaatgggtagc aaaaggaaaa aaaaatatat ttggacaa.ca 18O agtaaaagta atggaaatgc aaagtgaagc tiggagcaagt ggagcagtac atggaagttt 24 O acaa.gctgga gcattalacta gtact tatac togcaagt caa goattatt at taatgat acc 3OO taatatgt at aaaatagotg gagaattatt acctggagta titt catgitaa gtgcaagagc 360 US 2016/0160223 A1 Jun. 9, 2016 19

- Continued agtagcagca aatagtttaa atatatttgg agat catcaa gatgtaatgg caact agaca 42O aactggattit gcattatttg Cagaaagtag titacaacaa gtaatggatt taagtgcagt 48O agcacattta agtgcaataa aaggaagagt accttttgta aattitttittg atggatttag 54 O aactagt cat gaaatacaaa aaatagaatt attagaatat gaagaattag caaaattagt 6OO agat caaaaa gcattaaatg attittagaag aagagcatta aatcc tigatc atcct gtaac 660 tagaggaact gcacaaaatc ctdatatata ttittcaagaa agagaagitaa gtaatactta 72 O titatgaagica ttacctgaaa tagtagaagg atatatgcaa gaaatgacta aattaactgg 78O aagagaatat catttattta attattatgg agcaaaagat gcagaaagaa taataatagc 84 O aatgggaagt gitatgtgaaa citgtagaaga agtaatagat tatttalacta Ctalaaggaga 9 OO aaaagtagga ttattaactg tacatttata tag accttitt agtataaaac attittatgaa 96.O atatatacct aaaactgtaa aaaaaatago agtattagat agaactaaag aacctggaag O2O tataggagaa cctittatatt tagatgtaaa aaatgcttitt tatggacaag aagtaca acc O8O tgtaat agtt ggaggaagat atggattagg atctaaagat gtattacct a gtcat at att 14 O acct g tattt gaaaatttaa aaagtgataa acctaaagat agatt tactt taagtatagt 2OO agatgatgta actaatact a gtttacctgt tdgagaagat ataaatact a citcctgaagg 26 O aact actgca ttaaattitt ggggattagg aagtgatgga actgttggag caaataaatc 32O tgcaataaaa at aataggag at Catactga tatgt atgca Caaggatatt ttgct tatga 380 tagtaaaaaa agtggaggaa taactgtaag ticatttalaga tittggaaaaa gtc.ctataaa 44 O aagt cott at ttaatagata aag cagattt tdtag cagta cataatcaaa gttatgttca SOO taaatatgat g tattagcag gattaaaaaa aggaggaaat tttitt attaa atact.gitatg 560 gact caagaa gaattagaaa aagaattacc togcaagtatgaaaaagtata tagcaaataa 62O tgatataaag ttittatactt taaatgcagt aaaaatagoa caagaaatag gattaggagg 68O aagaataaat atgatatgtc. aaagtgcatt ttittaaaata gcaaatataa tacctataga 74 O tgatgcagta aaatatttaa aagaa.gcagt agtaact agt tatggaaaaa aaggacaaaa 8OO agttgtagat atgaataatg cagcaataga taaaggagta aatgcagtag taaaaataga 86 O tgtacctgca agttggaaag atgctgaaga tigaag cagda gcaactaaag aattacctaa 92 O atttatagaa aaaatagitaa atcc tatgaa tag acaagaa goagataaat tacct gtaag 98 O tgcatttgta ggaatggaag atggalactitt t cctgctgga actgcagctt atgaaaaaag 2O4. O aggaat agca ataaatgtac Citgaatggca agtagataaa titat acaat gtaat caatg 21OO tagttttgta tdtcct catg cagcaataag acctg tatta act actgaag aagaattagc 216 O taaagc acct Caaggatttg aagctaaaga tigcaaatgga gctaaaggat taaaatttac 222 O tatggcaata agt cctittag attgtagtgg atgtggaaat titgaagatg tatgtc.ctgc 228O aaaagaaaaa gcattagtaa toga aacctgt agatact caa ttaagtaaaa citgaag catg 234 O ggattatgca gtaaatgctg. tag cacataa agataatcct atgaaagata aatatagtgt 24 OO aaaa.gcaagt caatttgaac aacct ttatt agaatttagt ggagcatgtg Caggatgtgg 246 O agaaactic ct tatgtaaaat tagta actica attatttgga gatagaatga tigatagdaaa 252O tgcaactgga tigtag tagta tatggggagc aagtgct cot gcaactCctt at act actaa 2580 ttatagagga catggacct a gttgggcaaa ttctittattt galagataatg Cagaatatgg 264 O US 2016/0160223 A1 Jun. 9, 2016 20

- Continued attaggaatgtttittaggag taaaacaaac tagagaaaga ttacaagata aaatagalaga 27 OO agcattaaaa ggat.ctittaa gtgcagaatt aaaag.ca.gca tttgaagatt ggataaaaaa 276 O ttittgcagaa ggagaaggaa caa.gagaaag agcagataaa ataactgcat tattagaaaa 282O agaaaaagga tictaatgatt tattaaatga tatatatgaa aatagagatt ttittagtaaa 288O aagaagttcat tdataatag gaggagatgg atggggatat gat at aggat atggaggagt 294 O agat catgta ttagcaagta atgaagatgt aaatatatta gtattagata citgaagtata 3 OOO tagtaatact ggaggacaaa gtagtaaaag tactic ct act gcagcagtag caaaatttgc 3 O 6 O agcaagtgga aaaaaaacta aaaaaaaaga tittaggaatg atggcaatga gtt atggata 312 O tgtatatgta gcacaaataa gtatgggagc tigataaaaat caa.gctittaa aag caataca 318O tgaagctgaa gcatat catg gaccaagttt aataatagot tatgcaccitt gtataaatca 324 O tggattaaga gtaggaatgg gaaaaagt ca aagagaa.gca aaaagagcag tagattgttgg 33 OO at attgggca ttatatagat ataatcctga attaaaagaa gaaggaaaaa aatcttittag 3360 tittagatagt aaagaaccta c tact gattt taaagaattt ttaatgggag aagtaagata 342O tag tagttta gcaaaacaat titcctgat ca agcagatgct ttatttgaaa aaacaaaaaa 3480 agatgcatta caaagaatag ctggatataa aaaattagat aatgaacaat aagctago 3.538

<210s, SEQ ID NO 4 & 211 LENGTH 1170 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized Clostridium autoethanogenum LZ1561 pyruvate : ferredoxin oxidoreductase <4 OOs, SEQUENCE: 4 Met Arg Llys Met Lys Thr Met Asp Gly Asn. Thir Ala Ala Ala Tyr Ile 1. 5 1O 15 Ser Tyr Ala Phe Thr Asp Val Ala Ala Ile Tyr Pro Ile Thr Pro Ser 2O 25 3O Ser Pro Met Ala Glu. His Val Asp Glu Trp Val Ala Lys Gly Lys Llys 35 4 O 45 Asn Ile Phe Gly Glin Glin Val Llys Val Met Glu Met Glin Ser Glu Ala SO 55 6 O Gly Ala Ser Gly Ala Wal His Gly Ser Lieu. Glin Ala Gly Ala Lieu. Thir 65 70 7s 8O Ser Thr Tyr Thr Ala Ser Glin Gly Lieu. Leu Lleu Met Ile Pro Asn Met 85 90 95 Tyr Lys Ile Ala Gly Glu Lieu. Lieu Pro Gly Val Phe His Val Ser Ala 1OO 105 11 O

Arg Ala Val Ala Ala Asn. Ser Lieu. Asn. Ile Phe Gly Asp His Glin Asp 115 12 O 125

Val Met Ala Thr Arg Glin Thr Gly Phe Ala Leu Phe Ala Glu Ser Ser 13 O 135 14 O

Val Glin Glin Val Met Asp Lieu. Ser Ala Val Ala His Lieu. Ser Ala Ile 145 150 155 160

Lys Gly Arg Val Pro Phe Val Asin Phe Phe Asp Gly Phe Arg Thr Ser 1.65 17O 17s

His Glu Ile Glin Lys Ile Glu Lieu. Lieu. Glu Tyr Glu Glu Lieu Ala Lys US 2016/0160223 A1 Jun. 9, 2016 21

- Continued

18O 185 19 O Lieu Val Asp Gln Lys Ala Lieu. Asn Asp Phe Arg Arg Arg Ala Lieu. Asn 195 2OO 2O5 Pro Asp His Pro Val Thr Arg Gly Thr Ala Glin Asn Pro Asp Ile Tyr 21 O 215 22O Phe Glin Glu Arg Glu Val Ser Asn Thr Tyr Tyr Glu Ala Leu Pro Glu 225 23 O 235 24 O Ile Val Glu Gly Tyr Met Glin Glu Met Thr Lys Lieu. Thr Gly Arg Glu 245 250 255 Tyr His Lieu. Phe Asn Tyr Tyr Gly Ala Lys Asp Ala Glu Arg Ile Ile 26 O 265 27 O Ile Ala Met Gly Ser Val Cys Glu Thr Val Glu Glu Val Ile Asp Tyr 27s 28O 285 Lieu. Thir Thr Lys Gly Glu Lys Val Gly Lieu. Lieu. Thr Val His Lieu. Tyr 29 O 295 3 OO Arg Pro Phe Ser Ile Llys His Phe Met Lys Tyr Ile Pro Llys Thr Val 3. OS 310 315 32O Llys Lys Ile Ala Val Lieu. Asp Arg Thr Lys Glu Pro Gly Ser Ile Gly 3.25 330 335 Glu Pro Lieu. Tyr Lieu. Asp Wall Lys Asn Ala Phe Tyr Gly Glin Glu Val 34 O 345 35. O Glin Pro Val Ile Val Gly Gly Arg Tyr Gly Lieu. Gly Ser Lys Asp Wall 355 360 365 Lieu Pro Ser His Ile Lieu Pro Val Phe Glu Asn Lieu Lys Ser Asp Llys 37 O 375 38O Pro Lys Asp Arg Phe Thr Lieu. Ser Ile Val Asp Asp Val Thir Asn Thr 385 390 395 4 OO Ser Leu Pro Val Gly Glu Asp Ile Asn. Thir Thr Pro Glu Gly. Thir Thr 4 OS 41O 415 Ala Cys Llys Phe Trp Gly Lieu. Gly Ser Asp Gly Thr Val Gly Ala Asn 42O 425 43 O Llys Ser Ala Ile Lys Ile Ile Gly Asp His Thr Asp Met Tyr Ala Glin 435 44 O 445 Gly Tyr Phe Ala Tyr Asp Ser Lys Llys Ser Gly Gly Ile Thr Val Ser 450 45.5 460 His Lieu. Arg Phe Gly Llys Ser Pro Ile Llys Ser Pro Tyr Lieu. Ile Asp 465 470 47s 48O Lys Ala Asp Phe Val Ala Wal His Asn Glin Ser Tyr Val His Llys Tyr 485 490 495 Asp Val Lieu Ala Gly Lieu Lys Lys Gly Gly Asn. Phe Lieu. Lieu. Asn Thr SOO 505 51O

Val Trp Thr Glin Glu Glu Lieu. Glu Lys Glu Lieu Pro Ala Ser Met Lys 515 52O 525

Llys Tyr Ile Ala Asn. Asn Asp Ile Llys Phe Tyr Thr Lieu. Asn Ala Val 53 O 535 54 O

Lys Ile Ala Glin Glu Ile Gly Lieu. Gly Gly Arg Ile Asn Met Ile Cys 5.45 550 555 560

Glin Ser Ala Phe Phe Lys Ile Ala Asn. Ile Ile Pro Ile Asp Asp Ala 565 st O sts

Val Llys Tyr Lieu Lys Glu Ala Val Val Thir Ser Tyr Gly Lys Lys Gly 58O 585 59 O US 2016/0160223 A1 Jun. 9, 2016 22

- Continued

Glin Llys Val Val Asp Met Asn. Asn Ala Ala Ile Asp Llys Gly Val Asn 595 6OO 605 Ala Val Val Lys Ile Asp Val Pro Ala Ser Trp Lys Asp Ala Glu Asp 610 615 62O Glu Ala Ala Ala Thr Lys Glu Lieu Pro Llys Phe Ile Glu Lys Ile Val 625 630 635 64 O Asn Pro Met Asn Arg Glin Glu Gly Asp Llys Lieu Pro Val Ser Ala Phe 645 650 655 Val Gly Met Glu Asp Gly Thr Phe Pro Ala Gly Thr Ala Ala Tyr Glu 660 665 67 O Lys Arg Gly Ile Ala Ile Asn Val Pro Glu Trp Glin Val Asp Llys Cys 675 68O 685 Ile Glin Cys Asn Glin Cys Ser Phe Val Cys Pro His Ala Ala Ile Arg 69 O. 695 7 OO Pro Val Lieu. Thir Thr Glu Glu Glu Lieu Ala Lys Ala Pro Glin Gly Phe 7 Os 71O 71s 72O Glu Ala Lys Asp Ala Asn Gly Ala Lys Gly Lieu Lys Phe Thr Met Ala 72 73 O 73 Ile Ser Pro Lieu. Asp Cys Ser Gly Cys Gly Asn. Cys Glu Asp Val Cys 740 74. 7 O Pro Ala Lys Glu Lys Ala Lieu Val Met Llys Pro Val Asp Thr Glin Lieu. 7ss 760 765 Ser Lys Thr Glu Ala Trp Asp Tyr Ala Val Asn Ala Val Ala His Lys 770 775 78O Asp ASn Pro Met Lys Asp Llys Tyr Ser Val Lys Ala Ser Glin Phe Glu 78s 79 O 79. 8OO Glin Pro Lieu. Lieu. Glu Phe Ser Gly Ala Cys Ala Gly Cys Gly Glu Thir 805 810 815 Pro Tyr Val Lys Lieu Val Thr Gln Leu Phe Gly Asp Arg Met Met Ile 82O 825 83 O Ala Asn Ala Thr Gly Cys Ser Ser Ile Trp Gly Ala Ser Ala Pro Ala 835 84 O 845 Thr Pro Tyr Thr Thr Asn Tyr Arg Gly His Gly Pro Ser Trp Ala Asn 850 855 860 Ser Lieu. Phe Glu Asp Asn Ala Glu Tyr Gly Lieu. Gly Met Phe Lieu. Gly 865 87O 87s 88O Val Lys Glin Thr Arg Glu Arg Lieu. Glin Asp Llys Ile Glu Glu Ala Lieu 885 890 895 Lys Gly Ser Lieu. Ser Ala Glu Lieu Lys Ala Ala Phe Glu Asp Trp Ile 9 OO 905 91 O Lys Asn. Phe Ala Glu Gly Glu Gly Thr Arg Glu Arg Ala Asp Llys Ile 915 92 O 925

Thir Ala Lieu. Lieu. Glu Lys Glu Lys Gly Ser Asn Asp Lieu. Lieu. Asn Asp 93 O 935 94 O Ile Tyr Glu Asn Arg Asp Phe Lieu Val Lys Arg Ser His Trp Ile Ile 945 950 955 96.O Gly Gly Asp Gly Trp Gly Tyr Asp Ile Gly Tyr Gly Gly Val Asp His 965 97O 97.

Val Lieu Ala Ser Asn. Glu Asp Val Asn. Ile Lieu Val Lieu. Asp Thr Glu 98O 985 99 O US 2016/0160223 A1 Jun. 9, 2016 23

- Continued

Val Tyr Ser Asn Thr Gly Gly Glin Ser Ser Lys Ser Thr Pro Thr Ala 995 1OOO 1005 Ala Val Ala Llys Phe Ala Ala Ser Gly Lys Llys Thr Lys Llys Llys O1O O15 O2O Asp Leu Gly Met Met Ala Met Ser Tyr Gly Tyr Val Tyr Val Ala O25 O3 O O35 Glin Ile Ser Met Gly Ala Asp Lys Asn Glin Ala Lieu Lys Ala Ile O4 O O45 OSO His Glu Ala Glu Ala Tyr His Gly Pro Ser Lieu. Ile Ile Ala Tyr O55 O6 O O65 Ala Pro Cys Ile Asn His Gly Lieu. Arg Val Gly Met Gly Llys Ser Of O O7 O8O Glin Arg Glu Ala Lys Arg Ala Val Asp Cys Gly Tyr Trp Ala Lieu.

Tyr Arg Tyr Asn Pro Glu Lieu Lys Glu Glu Gly Llys Llys Ser Phe

Ser Lieu. Asp Ser Lys Glu Pro Thr Thr Asp Phe Lys Glu Phe Lieu.

Met Gly Glu Val Arg Tyr Ser Ser Lieu Ala Lys Glin Phe Pro Asp

Glin Ala Asp Ala Lieu. Phe Glu Lys Thir Lys Lys Asp Ala Lieu. Glin

Arg Ile Ala Gly Tyr Llys Llys Lieu. Asp ASn Glu Gln 16 O 65 17 O

<210s, SEQ ID NO 5 &211s LENGTH: 3725 &212s. TYPE: DNA <213> ORGANISM: Desulfovibrio africanus

<4 OOs, SEQUENCE: 5 tctagataag gaggtoggac atgggaaaaa aaatgatgac tactgatgga aatactgcaa. 6 O ctgcacatgt agctitatgca atgagtgaag tag cagdaat at atcctata act cotagta 12 O gtactatggg agaagaagca gacgattggg cagcacaggg aagaaaaaat at atttggac 18O aaactittaac tataagagaa atgcaaagtg aagctggtgc tigctggtgca gtacatggtg 24 O cattagcago toggtgcatta act act actt ttact.gcaa.g. tcagggatta t tact tatga 3OO tacctaatat gtacaaaata agtggtgaat tattacctgg togt attt cat gtaactgcaa. 360 gagcaatago agcacatgca cittagtatat ttggagat catcaagatata tatgcagcaa. 42O gacagactgg atttgcaatgttagcaagta gtagttaca agaggcacat gatatggcac 48O ttgtag caca tttagcagca atagaaagta atgtacctitt tatgcatttt tttgatggat 54 O ttagaactag to atgaaata caaaaaatag aag tattaga titatgcagat atggcaagtt 6OO tagtaaatca aaaagcatta gcagaattta gagcaaaaag tatgaatcct gaa catcctic 660 atgtaagagg aactgcacaa aatcCtgata t at attittca gggaagagala gcagcaaatc 72 O cittatt atct taaagtacct ggaatagtag ctgaatatat gcaaaaagta gcaagtttaa 78O

Ctggaagaag titataaactt tttgattatgttggagcacc tatgcagala agggitaatag 84 O talagtatggg aagtagttgt gaalactatag aagaagtaat aaatcatctt gcagcaaaag 9 OO gtgaaaaaat agg acttata aaagtaagac tittatagacc titttgtaagt gaagcattitt 96.O US 2016/0160223 A1 Jun. 9, 2016 24

- Continued ttgcagcatt acctgcaagt gcaaaagtaa taact g tatt agatagaact aaagaac citg O2O gtgcacctgg tdatcCttta t at Cttgatg tatgtag tec atttgtagala aggggtgaag O8O

Caatgcctaa aat acttgct ggaagatatggattaggaag taaagaattt agt cctgcaa. 14 O tggtaaaaag titatatgat aatatgagtg gtgcaaaaaa aaatcattt C actgtaggaa 2OO tagaagatga tigtaactgga act agtttac ctdtagataa togcatttgca gatact actic 26 O Ctaaagg tac tatacaatgt caattittggg gaCttggagc agatggaact gttggagcaa. 32O ataaacaa.gc aataaaaata attggagata atact gattt atttgcacaa goatattitta 38O gttatgatag taaaaaaagt ggtggaataa ctataagtica tttalagattt ggagaaaaac 44 O ctatacaaag tact tattta gtaaatagag cagattatgt agcatgtcat aatcc tigctt SOO atgtaggaat atatgatata ttagalaggta taaaagatgg toggaacttitt gtact taata 560 gtcCttggag tagtttagaa gatatggata alacattt acc tagtggaata aaaagaact a 62O tagcaaataa gaaattaaaa ttittataata tagatgcagt aaaaatagda actgatgtag 68O gattaggtgg aagaataaat atgataatgc aaactgcatt ttittaaatta gctggtgitat 74 O taccttittga aaaag.cagta gattt attaa aaaaaagtat acataaagct tatggaaaaa 8OO alaggtgaaaa aattgtaaaa atgaatactg atgcagtaga t caag cagta act agtttac 86 O aagaatttaa at atcc tdat agttggaaag atgcaccago agaaactaaa goaga accta 92 O tgactaatga atttitttaaa aatgtagtaa aacctatatt aacticaacaa gotgataaat 98 O tacctgitatic togcatttgaa goagatggaa gattt cotct toggaactagt caatttgaaa 2O4. O aaagaggtgt agctataaac gtacct cagt gggttcCtga aaattgtata cagtgtaatc 21OO aatgtgcatt totatgtc.ct catagtgcaa tact tccagt attagcaaaa gaagaagaat 216 O tagttggagc accagcaa at titt actgcac ttgaa.gcaaa gggaaaagaa cittaaaggat 222 O ataaatttag aatacagata aatactittag attgtatggg atgtggaaat tdtgcagata 228O tatgtc.ct co taaagaaaaa goacttgtaa tdcagoctot tdatact caa agagatgcac 234 O aagtacctaa tittagaatat gcagotagaa tacct gtaaa aagtgaagta ttacctagag 24 OO at agtttaaa aggat.cticaa tittcaagaac ctittaatgga atttagtggit gcatgtag td 246 O gatgtggtga aacticcittat gtaagagtaa taact caatt atttggagaa agaatgttta 252O tagcaaatgc aactggatgt agtagtatat ggggagcaag tic acct agt atgccttata 2580 aalactaatag acttggacaa ggacctgcat ggggaaatag titt atttgaa gatgcagcag 264 O aatatggatt taatgaat atgagtatgt ttgcaagaag aact cattta gcagatttag 27 OO

Ctgcaaaagc attagaaagt gatgcaagtg gtgatgtaaa agaag catta Caaggttggit 276 O tagctggaaa aaatgatcct ataaaaagta aagaatatgg togataaactt aaaaaactitt 282O tagcaggaca aaaagatgga t tatt aggac aaataggagc aatgagtgat Ctttatact a 288O aaaaaagtgt atggat attt ggaggtgatg gatgggctta tatatagga tatggtggac 294 O ttgat catgt acttgcatct ggtgaagatg taaatgitatt totaatggat actgaagitat 3 OOO at agtaatac tdtggacaa agtag taaag caact Cotac tigtgcagta gcaaaatttg 3 O 6 O

Cagcagctgg aaaaagaact ggaaaaaaag atttagcaag aatggtaatg act tatggat 312 O atgtatatgt agcaactgta t citatgggat at agtaalaca acaattt citt aaagtattaa 318O aagaagicaga aagtttitcct ggacctagtt tagtaatago titatgctact totataaatc 324 O US 2016/0160223 A1 Jun. 9, 2016 25

- Continued aaggattaag aaaagg tatg ggaaaaagtic aagatgtaat gaatactgca gtaaaaagtg 33 OO gatattggcc tittatttaga tatgatcc ta gacttgcago toagggaaaa aatcc ttitt c 3360 aattagatag taalagcacct gatggaagtg tagaagaatt tot tatggca caaaatagat 342O ttgcagtact tatagaagt titt Cotgaag atgcaaaaag attalaga.gca Caagtag cac 3480 atgaattaga tigtaagattit aaagaattag alacacatggc agcaactaat at atttgaaa 354 O gttittgcacc agc.cggtggit aaa.gcagatg gatctgtaga titttggaga a ggtgcagaat 36OO tttgtact ag agatgatact cct atgatgg caaga cctga tagtggtgaa gcatgtgat C 366 O aaaatagagc tiggaactagt gaacaac agg gtgat ct tag taaaagaact aaaaaataag 372 O ctago 3725

<210s, SEQ ID NO 6 &211s LENGTH: 1232 212. TYPE: PRT <213> ORGANISM: Desulfovibrio africanus

<4 OOs, SEQUENCE: 6 Met Gly Lys Llys Met Met Thir Thr Asp Gly Asn Thr Ala Thr Ala His 1. 5 1O 15 Val Ala Tyr Ala Met Ser Glu Val Ala Ala Ile Tyr Pro Ile Thr Pro 2O 25 3O Ser Ser Thr Met Gly Glu Glu Ala Asp Asp Trp Ala Ala Glin Gly Arg 35 4 O 45 Lys Asn Ile Phe Gly Glin Thr Lieu. Thir Ile Arg Glu Met Glin Ser Glu SO 55 6 O Ala Gly Ala Ala Gly Ala Wal His Gly Ala Lieu Ala Ala Gly Ala Lieu 65 70 7s 8O Thir Thir Thr Phe Thr Ala Ser Glin Gly Lieu. Leu Lleu Met Ile Pro Asn 85 90 95 Met Tyr Lys Ile Ser Gly Glu Lieu. Leu Pro Gly Val Phe His Val Thr 1OO 105 11 O Ala Arg Ala Ile Ala Ala His Ala Lieu. Ser Ile Phe Gly Asp His Glin 115 12 O 125 Asp Ile Tyr Ala Ala Arg Glin Thr Gly Phe Ala Met Lieu Ala Ser Ser 13 O 135 14 O Ser Val Glin Glu Ala His Asp Met Ala Lieu Val Ala His Lieu Ala Ala 145 150 155 160 Ile Glu Ser Asn Val Pro Phe Met His Phe Phe Asp Gly Phe Arg Thr 1.65 17O 17s Ser His Glu Ile Glin Lys Ile Glu Val Lieu. Asp Tyr Ala Asp Met Ala 18O 185 19 O

Ser Lieu Val Asin Glin Lys Ala Lieu Ala Glu Phe Arg Ala Lys Ser Met 195 2OO 2O5

Asn Pro Glu. His Pro His Val Arg Gly Thr Ala Glin Asn Pro Asp Ile 21 O 215 22O

Tyr Phe Glin Gly Arg Glu Ala Ala Asn Pro Tyr Tyr Lieu Lys Val Pro 225 23 O 235 24 O

Gly Ile Val Ala Glu Tyr Met Glin Llys Val Ala Ser Lieu. Thr Gly Arg 245 250 255

Ser Tyr Lys Lieu. Phe Asp Tyr Val Gly Ala Pro Asp Ala Glu Arg Val 26 O 265 27 O US 2016/0160223 A1 Jun. 9, 2016 26

- Continued

Ile Wall Ser Met Gly Ser Ser Cys Glu Thir Ile Glu Glu Wall Ile Asn 27s 285

His Luell Ala Ala Gly Glu Ile Gly Luell Ile Wall Arg Luell 29 O 295 3 OO

Tyr Arg Pro Phe Wall Ser Glu Ala Phe Phe Ala Ala Lell Pro Ala Ser 3. OS 310 315

Ala Wall Ile Thir Wall Lell Asp Arg Thir Glu Pro Gly Ala Pro 3.25 330 335

Gly Asp Pro Luell Tyr Lell Asp Wall Cys Ser Ala Phe Wall Glu Arg Gly 34 O 345 35. O

Glu Ala Met Pro Ile Lell Ala Gly Arg Gly Lell Gly Ser 355 360 365

Glu Phe Ser Pro Ala Met Wall Ser Wall Asp Asn Met Ser Gly 37 O 375

Ala Asn His Phe Thir Wall Gly Ile Glu Asp Asp Wall Thir Gly 385 390 395 4 OO

Thir Ser Luell Pro Wall Asp Asn Ala Phe Ala Asp Thir Thir Pro Lys Gly 4 OS 415

Thir Ile Glin Cys Glin Phe Trp Gly Luell Gly Ala Asp Gly Thir Wall Gly 425 43 O

Ala Asn Lys Glin Ala Ile Ile Ile Gly Asp Asn Thir Asp Luell Phe 435 44 O 445

Ala Glin Gly Tyr Phe Ser Tyr Asp Ser Ser Gly Gly Ile Thir 450 45.5 460

Ile Ser His Luell Arg Phe Gly Glu Pro Ile Glin Ser Thir Luell 465 470

Wall Asn Arg Ala Asp Wall Ala His ASn Pro Ala Wall Gly 485 490 495

Ile Asp Ile Lell Glu Gly Ile Lys Asp Gly Gly Thir Phe Wall Luell SOO 505 51O

Asn Ser Pro Trp Ser Ser Lell Glu Asp Met Asp His Luell Pro Ser 515 525

Gly Ile Arg Thir Ile Ala Asn Luell Lys Phe Asn Ile 53 O 535 54 O

Asp Ala Wall Ile Ala Thir Asp Wall Gly Luell Gly Gly Arg Ile Asn 5.45 550 555 560

Met Ile Met Glin Thir Ala Phe Phe Luell Ala Gly Wall Luell Pro Phe 565 st O sts

Glu Ala Wall Asp Lell Lell Lys Ser Ile His Ala Gly 58O 585 59 O

Gly Glu Ile Wall Lys Met Asn Thir Asp Ala Wall Glin 595 605

Ala Wall Thir Ser Lell Glin Glu Phe Pro Asp Ser Trp Asp 610 615 62O

Ala Pro Ala Glu Thir Lys Ala Glu Pro Met Thir Asn Glu Phe Phe Lys 625 630 635 64 O

Asn Wall Wall Pro Ile Lell Thir Glin Glin Gly Asp Luell Pro Wall 645 650 655

Ser Ala Phe Glu Ala Asp Gly Arg Phe Pro Luell Gly Thir Ser Glin Phe 660 665 67 O US 2016/0160223 A1 Jun. 9, 2016 27

- Continued

Glu Lys Arg Gly Val Ala Ile Asin Val Pro Glin Trp Val Pro Glu Asn 675 68O 685 Cys Ile Glin Cys Asn Gln Cys Ala Phe Val Cys Pro His Ser Ala Ile 69 O. 695 7 OO Lieu Pro Val Lieu Ala Lys Glu Glu Glu Lieu Val Gly Ala Pro Ala Asn 7 Os 71O 71s 72O Phe Thr Ala Lieu. Glu Ala Lys Gly Lys Glu Lieu Lys Gly Tyr Llys Phe 72 73 O 73 Arg Ile Glin Ile Asn. Thir Lieu. Asp Cys Met Gly Cys Gly Asn. Cys Ala 740 74. 7 O Asp Ile Cys Pro Pro Llys Glu Lys Ala Lieu Val Met Gln Pro Lieu. Asp 7ss 760 765 Thr Glin Arg Asp Ala Glin Val Pro Asn Lieu. Glu Tyr Ala Ala Arg Ile 770 775 78O Pro Val Lys Ser Glu Val Lieu Pro Arg Asp Ser Lieu Lys Gly Ser Glin 78s 79 O 79. 8OO Phe Glin Glu Pro Leu Met Glu Phe Ser Gly Ala Cys Ser Gly Cys Gly 805 810 815 Glu Thr Pro Tyr Val Arg Val Ile Thr Glin Leu Phe Gly Glu Arg Met 82O 825 83 O Phe Ile Ala Asn Ala Thr Gly Cys Ser Ser Ile Trp Gly Ala Ser Ala 835 84 O 845 Pro Ser Met Pro Tyr Llys Thr ASn Arg Lieu. Gly Glin Gly Pro Ala Trp 850 855 860 Gly Asn. Ser Lieu. Phe Glu Asp Ala Ala Glu Tyr Gly Phe Gly Met Asn 865 87O 87s 88O Met Ser Met Phe Ala Arg Arg Thr His Lieu Ala Asp Lieu Ala Ala Lys 885 890 895 Ala Lieu. Glu Ser Asp Ala Ser Gly Asp Wall Lys Glu Ala Lieu. Glin Gly 9 OO 905 91 O Trp Lieu Ala Gly Lys Asn Asp Pro Ile Llys Ser Lys Glu Tyr Gly Asp 915 92 O 925 Llys Lieu Lys Llys Lieu. Lieu Ala Gly Glin Lys Asp Gly Lieu. Lieu. Gly Glin 93 O 935 94 O Ile Ala Ala Met Ser Asp Leu Tyr Thr Lys Llys Ser Val Trp Ile Phe 945 950 955 96.O Gly Gly Asp Gly Trp Ala Tyr Asp Ile Gly Tyr Gly Gly Lieu. Asp His 965 97O 97. Val Lieu Ala Ser Gly Glu Asp Val Asn Val Phe Wal Met Asp Thr Glu 98O 985 99 O Val Tyr Ser Asn Thr Gly Gly Glin Ser Ser Lys Ala Thr Pro Thr Gly 995 1OOO 1005

Ala Val Ala Llys Phe Ala Ala Ala Gly Lys Arg Thr Gly Llys Llys O1O 1015 O2O

Asp Leu Ala Arg Met Val Met Thr Tyr Gly Tyr Val Tyr Val Ala O25 1O3 O O35

Thr Val Ser Met Gly Tyr Ser Lys Glin Glin Phe Leu Lys Val Lieu. O4 O 1045 OSO

Lys Glu Ala Glu Ser Phe Pro Gly Pro Ser Lieu Val Ile Ala Tyr O55 106 O O65

Ala Thr Cys Ile Asin Glin Gly Lieu. Arg Lys Gly Met Gly Llys Ser US 2016/0160223 A1 Jun. 9, 2016 28

- Continued

Gln Asp Val Met Asn Thr Ala Val Lys Ser Gly Tyr Trp Pro Leu O85 O9 O O95 Phe Arg Tyr Asp Pro Arg Lieu Ala Ala Glin Gly Lys Asn Pro Phe OO O5 10 Glin Lieu. Asp Ser Lys Ala Pro Asp Gly Ser Val Glu Glu Phe Lieu.

Met Ala Glin Asn Arg Phe Ala Val Lieu. Asp Arg Ser Phe Pro Glu

Asp Ala Lys Arg Lieu. Arg Ala Glin Val Ala His Glu Lieu. Asp Wall

Arg Phe Lys Glu Lieu. Glu. His Met Ala Ala Thr Asn. Ile Phe Glu

Ser Phe Ala Pro Ala Gly Gly Lys Ala Asp Gly Ser Val Asp Phe 7s 8O 85 Gly Glu Gly Ala Glu Phe Cys Thr Arg Asp Asp Thr Pro Met Met 90 95 2OO Ala Arg Pro Asp Ser Gly Glu Ala Cys Asp Glin Asn Arg Ala Gly 2O5 21 O 215 Thir Ser Glu Glin Glin Gly Asp Lieu. Ser Lys Arg Thr Lys Llys 22O 225 23 O

<210 SEQ ID NO 7 &211s LENGTH: 1617 &212s. TYPE: DNA <213> ORGANISM: Clostridium autoethanogenum LZ1561 <4 OO > SEQUENCE: 7 atggittatga aagctgctga agcagttatc caatgtttaa aaaaagaaaa totaaatatg 6 O gtatttgggt atcctggtgc tigcagtggitt CCtatatatg aagctttgag aaaat cagat 12 O gtgaag cata tattagtaag acaggaacaa gCtgcaggac actictgctag tatatgct 18O aggit caactg gagaagttgg agtctgtata gttacat cag gacctggcgc alactaatcta 24 O attact.gc.ca ttgctgctgc atatatggat tcc attcctic ttgttgttat tacgggt cag 3OO gttalagtota Cattaatcgg aagggatgta titcCaagaat tagatat cac aggtgctaca 360 gaat cittitta caaaatataa ttittcttgta agagatgcta aatctatacc taagactata 42O aaggaagc at tittatatagc tigaaactggit agaaaaggcc ctgtgcttgt agatatacct 48O atggatataa tigaagaaga tattgattitt gaatat cct aaagtgtaala tataagagga 54 O tacaaaccta citgttaaagg acact ctdgg caaataaaga aaataataga tagaatcaaa 6OO gttagcaaga gacct Ctt at ttgttgcaggt ggcggagitta tactggcaaa to acaaaaa 660 gaactggagc aatttgttaa aaaat cacat atacctgttgttcat actict tatgggaaaa 72 O ggatgtataa atgaaaatag togattatt at gtaggitttaa tagg tactica toggctittgct 78O tatgcc-aata aagttgtaca aaatgcagat gtactaatac ttattggagc tagagct tca 84 O gatagaactg. t cagtggagt aaaaagttitt gcaaaggatg Cagatataat it catatagat 9 OO atcgatcc td citgaaatagg taaaattctgaac acttata titc.ca.gtggit tdgtgattgt 96.O ggaagtgttt tat cqgattt aaataaagaa at agtagct C Cacagacaga aaaatggatg 1 O2O gaagaaatta aaaattggala aaaagatttg tatatagaaa gaaagcctac agataaagtt 108 O US 2016/0160223 A1 Jun. 9, 2016 29

- Continued aatcc.caaat atgtgttaaa aactgtttct gatacattag gagaagaggit tattittgaca 14 O gCagatgtag gacaaaatca gttgttggtgt gcc.cgtaatt ttaggatgac agggaataga 2OO aagtttittaa cittctggagg cct cqgaact atgggatatt ct citcc.cagc agctattggit 26 O gctaaaattig catgtc.ctga taa.gcaagtt at agcttittg Caggtgatgg toggatttcaa 32O atgagt ctitt ttgaacttgg aactattgcc gaaaataatc taaac attat tatagittittg 38O tittaacaact Caggactggg tatggittagg gagatacaag acaataaata ttctggtgaa 44 O tittggagtaa attittaggac caatccagat tttgtaaaac ttgcagaagic ctatoggatta SOO aaagctaaga gagtagaaaa tatt ctgaa tittaacggag tittittagaga agcattagat 560 t caa.gcaagg catttittaat agagtgcatt gtagatcctic atgagagaac tttittag 617

<210s, SEQ ID NO 8 &211s LENGTH: 538 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum LZ1561

<4 OOs, SEQUENCE: 8 Met Val Met Lys Ala Ala Glu Ala Val Ile Glin Cys Lieu Lys Lys Glu 1. 5 1O 15 Asn Val Asn Met Val Phe Gly Tyr Pro Gly Ala Ala Val Val Pro Ile 2O 25 3O Tyr Glu Ala Lieu. Arg Llys Ser Asp Wall Lys His Ile Lieu Val Arg Glin 35 4 O 45 Glu Glin Ala Ala Gly His Ser Ala Ser Gly Tyr Ala Arg Ser Thr Gly SO 55 6 O Glu Val Gly Val Cys Ile Val Thr Ser Gly Pro Gly Ala Thr Asn Lieu. 65 70 7s 8O Ile Thr Ala Ile Ala Ala Ala Tyr Met Asp Ser Ile Pro Lieu Val Val 85 90 95 Ile Thr Gly Glin Val Lys Ser Thr Lieu. Ile Gly Arg Asp Val Phe Glin 1OO 105 11 O Glu Lieu. Asp Ile Thr Gly Ala Thr Glu Ser Phe Thr Lys Tyr Asin Phe 115 12 O 125 Lieu Val Arg Asp Ala Lys Ser Ile Pro Llys Thir Ile Llys Glu Ala Phe 13 O 135 14 O Tyr Ile Ala Glu Thr Gly Arg Lys Gly Pro Val Lieu Val Asp Ile Pro 145 150 155 160 Met Asp Ile Met Glu Glu Asp Ile Asp Phe Glu Tyr Pro Glu Ser Val 1.65 17O 17s Asn Ile Arg Gly Tyr Lys Pro Thr Val Lys Gly His Ser Gly Glin Ile 18O 185 19 O Llys Lys Ile Ile Asp Arg Ile Llys Val Ser Lys Arg Pro Lieu. Ile Cys 195 2OO 2O5

Ala Gly Gly Gly Val Ile Lieu Ala Asn Ala Glin Lys Glu Lieu. Glu Glin 21 O 215 22O

Phe Val Lys Llys Ser His Ile Pro Val Val His Thr Lieu Met Gly Lys 225 23 O 235 24 O

Gly Cys Ile Asin Glu Asn. Ser Asp Tyr Tyr Val Gly Lieu. Ile Gly. Thir 245 250 255

His Gly Phe Ala Tyr Ala Asn Llys Val Val Glin Asn Ala Asp Val Lieu. 26 O 265 27 O US 2016/0160223 A1 Jun. 9, 2016 30

- Continued

Ile Luell Ile Gly Ala Arg Ala Ser Asp Arg Thr Wall Ser Gly Val Lys 28O 285

Ser Phe Ala Lys Asp Ala Asp Ile Ile His Ile Asp Ile Asp Pro Ala 29 O 295 3 OO

Glu Ile Gly Lys Ile Lieu. Asn Thr Tyr Ile Pro Wall Wall Gly Asp Cys 3. OS 310 315 32O

Gly Ser Wall Lieu. Ser Asp Lieu. Asn Lys Glu Ile Wall Ala Pro Gn. Thir 3.25 330 335

Glu Trp Met Glu Glu Ile Llys Asn Trp Llys Asp Luell Tyr Ile 34 O 345 35. O

Glu Arg Lys Pro Thr Asp Llys Val Asn Pro Llys Wall Luell Lys Thr 355 360 365

Wall Ser Asp Thir Lieu. Gly Glu Glu Wall Ile Lieu. Thir Ala Asp Val Gly 37 O 375 38O

Glin Asn Glin Lieu. Trp Cys Ala Arg Asin Phe Arg Met Thir Gly Asn Arg 385 390 395 4 OO

Phe Luell Thir Ser Gly Gly Lieu Gly Thr Met Gly Tyr Ser Leul Pro 4 OS 41O 415

Ala Ala Ile Gly Ala Lys Ile Ala Cys Pro Asp Glin Wall Ile Ala 425 43 O

Phe Ala Gly Asp Gly Gly Phe Glin Met Ser Lieu. Phe Glu Luell Gly Thr 435 44 O 445

Ile Ala Glu Asn Asn Lieu. Asn. Ile Ile Ile Wall Lell Phe Asn Asn. Ser 450 45.5 460

Gly Luell Gly Met Val Arg Glu Ile Glin Asp Asn Ser Gly Glu 465 470 47s 48O

Phe Gly Wall Asin Phe Arg Thr Asn Pro Asp Phe Wall Luell Ala Glu 485 490 495

Ala Gly Lieu Lys Ala Lys Arg Wall Glu Asn Asp Ser Glu Phe Asn SOO 505

Gly Wall Phe Arg Glu Ala Lieu. Asp Ser Ser Lys Ala Phe Luell Ile Glu 515 525

Ile Wall Asp Pro His Glu Arg Thir Phe 53 O 535

<210s, SEQ ID NO 9 &211s LENGTH: 1677 &212s. TYPE: DNA <213> ORGANISM: Clostridium auto ethanogenum LZ1561

<4 OOs, SEQUENCE: 9 atgaaaataa agggagctga agtact atta aaatgitatga tggagcaagg tgtagatact 6 O gt attcggat atc.cgggagg agctgttitta cct atttatg atgcactata tgctgctaag 12 O ggaaagataa Ctcacatlatc. gacitt cacat gaacaagggg ctgct catgc tgcagatgga 18O tatgcaagat Ctacaggaaa gg taggagtt gtaattgcaa Cat Cagggcc gggagct act 24 O aatacggitta cagcaattgc tacagctitat atggatt CC9 tacct attgt agtatttaca 3OO ggac aggttg cgagaagt ct tcttggaaag gattcttitt c aagaagtaaa tattaaagat 360 attactgcat CCatalactaa gaaaagttgc attgtagaaa agg tagagga tittagctgat actgtaagag aggcattt ca aattgcagtt agtggaagac Caggacctgt agtag tagat 48O US 2016/0160223 A1 Jun. 9, 2016 31

- Continued atacctaaag acgtacaatc agctgaagta gaatatgagc ctitttagaag taagctttct 54 O gaaattaaag aaaagaaata ttittaattta aatgagtatg gagacagttt aaataaggca 6OO atagatatga taaataggag tagagacct gtaatttatt Caggtggagg alactgtcaca 660 t caggagctic aaaatgaatt gatggaactt gtagaaaaaa tag attcacc aattacctgt 72 O t cact tatgg gaataggagc titt cocggga aacaatgaat attatatggg tatggttgga 78O atgcatggaa gcc.gttgctic aaattatgca gtaagtaatt gtgacittatt aatagctata 84 O ggagct aggt ttagtgatag ggittataagc aaggtaagtg CCtttgctcc aaaagcaaga 9 OO ataatacaca tag acattga C cctaaggag tittggcaaaa acgtggatat agatgtagca 96.O ataaaaggag atgtaaaaga ggtacttcaa aagattaatt gcaagttaga aaaggc.cgac O2O

Cacagggatt ggatggagaa aataaaacag taaaagtgaac agtgtga accttittaaa O8O gaatgitaa at taagtic ctaa gtttataatg gataccttgt ataatcttac aggaggagaa 14 O tgcataatta ctacagaagt toggccaaaat caaatttgga citgcacaata ttittaaattic 2OO ttaaagcc aa galacatttgt at Ctt Caggc ggacttggala Ctatgggctt C9g acttgga 26 O gcttctatag gag catct at ggg taatcca gggaaaaagg taataaatgt agc aggggat 32O gggagctitta aaatgaattic tacagagctt gct actgttg ccaaatataa gct coctatt 38O gtacaattgc titttaaataa togtgcatta ggcatggitat atcaatggca ggatatgttc 44 O tatggaaaga ggttittcaaa tacagaactt ggaccagatgttgattt cat gaalacttgga SOO gaag.cgitatg gtataaagac ttittaagata gaaga caata gcc aggtaga gaaatgctta 560 aaggaagctic ttgact taaa talacctgta attatagaat gtgatataga taggaaagaa 62O alagg tattt C C tattgtacct Cogggagcg gct at at cag atttagtaga agagtaa 677

<210s, SEQ ID NO 10 &211s LENGTH: 558 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum LZ1561 <4 OOs, SEQUENCE: 10 Met Lys Ile Lys Gly Ala Glu Val Lieu. Lieu Lys Cys Met Met Glu Glin 1. 5 1O 15 Gly Val Asp Thr Val Phe Gly Tyr Pro Gly Gly Ala Val Lieu Pro Ile 2O 25 3O Tyr Asp Ala Lieu. Tyr Ala Ala Lys Gly Lys Ile Thr His Ile Ser Thr 35 4 O 45 Ser His Glu Glin Gly Ala Ala His Ala Ala Asp Gly Tyr Ala Arg Ser SO 55 6 O Thr Gly Llys Val Gly Val Val Ile Ala Thr Ser Gly Pro Gly Ala Thr 65 70 7s 8O

Asn Thr Val Thr Ala Ile Ala Thr Ala Tyr Met Asp Ser Val Pro Ile 85 90 95

Val Val Phe Thr Gly Glin Val Ala Arg Ser Lieu. Lieu. Gly Lys Asp Ser 1OO 105 11 O

Phe Glin Glu Val Asn. Ile Lys Asp Ile Thir Ala Ser Ile Thir Lys Llys 115 12 O 125

Ser Cys Ile Val Glu Lys Val Glu Asp Lieu Ala Asp Thr Val Arg Glu 13 O 135 14 O

Ala Phe Glin Ile Ala Val Ser Gly Arg Pro Gly Pro Val Val Val Asp US 2016/0160223 A1 Jun. 9, 2016 32

- Continued

145 150 155 160

Ile Pro Asp Wall Glin Ser Ala Glu Wall Glu Tyr Glu Pro Phe Arg 1.65 17O 17s

Ser Luell Ser Glu Ile Glu Lys Lys Phe Asn Luell Asn Glu 18O 185 19 O

Gly Asp Ser Lell Asn Ala Ile Asp Met Ile Asn Arg Ser Glu 195

Arg Pro Wall Ile Tyr Ser Gly Gly Gly Thir Wall Thir Ser Gly Ala Glin 21 O 215 22O

Asn Glu Luell Met Glu Lell Wall Glu Ile Asp Ser Pro Ile Thir Cys 225 23 O 235 24 O

Ser Luell Met Gly Ile Gly Ala Phe Pro Gly ASn Asn Glu Tyr Met 245 250 255

Gly Met Wall Gly Met His Gly Ser Arg Ser Asn Tyr Ala Wall Ser 26 O 265 27 O

Asn Asp Luell Lell Ile Ala Ile Gly Ala Arg Phe Ser Asp Arg Wall 285

Ile Ser Wall Ser Ala Phe Ala Pro Ala Arg Ile Ile His Ile 29 O 295 3 OO

Asp Ile Asp Pro Glu Phe Gly Asn Wall Asp Ile Asp Wall Ala 3. OS 310 315

Ile Gly Asp Wall Glu Wall Luell Glin Lys Ile Asn Lys Luell 325 330 335

Glu Ala Asp His Arg Asp Trp Met Glu Lys Ile Glin Trp 34 O 345 35. O

Ser Glu Glin Glu Pro Phe Lys Glu Lell Ser Pro Phe 355 360 365

Ile Met Asp Thir Lell Asn Luell Thir Gly Gly Glu Ile Ile Thir 37 O 375

Thir Glu Wall Gly Glin Asn Glin Ile Trp Thir Ala Glin Tyr Phe Phe 385 390 395 4 OO

Lell Pro Arg Thir Phe Wall Ser Ser Gly Gly Lell Gly Thir Met Gly 4 OS 415

Phe Gly Luell Gly Ala Ser Ile Gly Ala Ser Met Gly Asn Pro Gly 425 43 O

Wall Ile Asn Wall Ala Gly Asp Gly Ser Phe Met Asn Ser Thir 435 44 O 445

Glu Luell Ala Thir Wall Ala Lys Luell Pro Ile Wall Glin Luell Luell 450 45.5 460

Lell Asn Asn Arg Ala Lell Gly Met Wall Glin Trp Glin Asp Met Phe 465 470 47s 48O

Gly Arg Phe Ser Asn Thir Glu Luell Gly Pro Asp Wall Asp Phe 485 490 495

Met Luell Gly Glu Ala Gly Ile Thir Phe Ile Glu Asp SOO 505

Asn Ser Glin Wall Glu Luell Glu Ala Lell Asp Luell Asn Glu 515 525

Pro Wall Ile Ile Glu Asp Ile Asp Arg Glu Wall Phe Pro 53 O 535 54 O

Ile Wall Pro Pro Gly Ala Ala Ile Ser Asp Luell Wall Glu Glu 5.45 550 555 US 2016/0160223 A1 Jun. 9, 2016 33

- Continued

<210s, SEQ ID NO 11 &211s LENGTH: 486 &212s. TYPE: DNA <213> ORGANISM: Clostridium autoethanogenum LZ1561 <4 OOs, SEQUENCE: 11 catatgagtg tacttgtaga aaat catagt ggtgt attaa gtaaagtagc aggattattt 6 O agtagaagag gatataac at t catagttta actgttggag taactggtga t c ctgaaata 12 O agtagaatga citatagtaag tattggagat gattatatgt ttgaacaaat atctaaa.ca.g 18O cittaataaat tdatagaagt aataaaagta atagaattaa atcctgatgc aagtgtatat 24 O agagaattaa gtc.ttataaa agtaagtgca gaaagtaata acaaactitct tataatggaa 3OO agtgtaaata Cttittagagg taaaatagta gatatgaatgaaaaaagtat gataatagaa 360 ataactggaa atgaaaaaaa aataagtgcatttatagaat taatgaaacc titatggaata 42O aaagaaataa taagaactgg attaactgca ttacaaagag gatcaaaatt agaagattaa 48O gagotc 486

<210s, SEQ ID NO 12 &211s LENGTH: 158 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum LZ1561 < 4 OO SEQUENCE: 12 Met Ser Val Lieu Val Glu Asn His Ser Gly Val Lieu. Ser Llys Val Ala 1. 5 1O 15 Gly Lieu. Phe Ser Arg Arg Gly Tyr Asn. Ile His Ser Lieu. Thr Val Gly 2O 25 3O Val Thr Gly Asp Pro Glu Ile Ser Arg Met Thir Ile Val Ser Ile Gly 35 4 O 45 Asp Asp Tyr Met Phe Glu Glin Ile Ser Lys Glin Lieu. Asn Llys Lieu. Ile SO 55 6 O Glu Val Ile Llys Val Ile Glu Lieu. Asn Pro Asp Ala Ser Val Tyr Arg 65 70 7s 8O Glu Lieu. Ser Lieu. Ile Llys Val Ser Ala Glu Ser Asn. Asn Llys Lieu. Lieu. 85 90 95 Ile Met Glu Ser Val Asn Thr Phe Arg Gly Lys Ile Val Asp Met Asn 1OO 105 11 O Glu Lys Ser Met Ile Ile Glu Ile Thr Gly Asn. Glu Lys Lys Ile Ser 115 12 O 125 Ala Phe Ile Glu Lieu Met Llys Pro Tyr Gly Ile Llys Glu Ile Ile Arg 13 O 135 14 O

Thr Gly Lieu. Thir Ala Lieu. Glin Arg Gly Ser Lys Lieu. Glu Asp 145 150 155

<210s, SEQ ID NO 13 &211s LENGTH: 486 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Mutant Clostridium autoethanogenum LZ1561 IlvN (G-10-D) acetolactate synthase

<4 OOs, SEQUENCE: 13 US 2016/0160223 A1 Jun. 9, 2016 34

- Continued catatgagtg tacttgtaga aaat catagt gatgt attaa gtaaagtagc aggattattt 6 O agtagaagag gatataac at t catagttta actgttggag taactggtga t c ctgaaata 12 O agtagaatga citatagtaag tattggagat gattatatgt ttgaacaaat atctaaa.ca.g 18O cittaataaat tdatagaagt aataaaagta atagaattaa atcctgatgc aagtgtatat 24 O agagaattaa gtc.ttataaa agtaagtgca gaaagtaata acaaactitct tataatggaa 3OO agtgtaaata Cttittagagg taaaatagta gatatgaatgaaaaaagtat gataatagaa 360 ataactggaa atgaaaaaaa aataagtgcatttatagaat taatgaaacc titatggaata 42O aaagaaataa taagaactgg attaactgca ttacaaagag gatcaaaatt agaagattaa 48O gagotc 486

<210s, SEQ ID NO 14 &211s LENGTH: 158 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Mutant Clostridium autoethanogenum LZ1561 IlvN (G-10-D) acetolactate synthase

<4 OOs, SEQUENCE: 14 Met Ser Val Lieu Val Glu Asn His Ser Asp Val Lieu. Ser Llys Val Ala 1. 5 1O 15 Gly Lieu. Phe Ser Arg Arg Gly Tyr Asn. Ile His Ser Lieu. Thr Val Gly 2O 25 30 Val Thr Gly Asp Pro Glu Ile Ser Arg Met Thir Ile Val Ser Ile Gly 35 4 O 45 Asp Asp Tyr Met Phe Glu Glin Ile Ser Lys Glin Lieu. Asn Llys Lieu. Ile SO 55 6 O Glu Val Ile Llys Val Ile Glu Lieu. Asn Pro Asp Ala Ser Val Tyr Arg 65 70 7s 8O Glu Lieu. Ser Lieu. Ile Llys Val Ser Ala Glu Ser Asn. Asn Llys Lieu. Lieu. 85 90 95 Ile Met Glu Ser Val Asn Thr Phe Arg Gly Lys Ile Val Asp Met Asn 1OO 105 11 O Glu Lys Ser Met Ile Ile Glu Ile Thr Gly Asn. Glu Lys Lys Ile Ser 115 12 O 125 Ala Phe Ile Glu Lieu Met Llys Pro Tyr Gly Ile Llys Glu Ile Ile Arg 13 O 135 14 O Thr Gly Lieu. Thir Ala Lieu. Glin Arg Gly Ser Lys Lieu. Glu Asp 145 150 155

<210s, SEQ ID NO 15 &211s LENGTH: 1671 &212s. TYPE: DNA <213> ORGANISM: Clostridium autoethanogenum LZ1561

<4 OOs, SEQUENCE: 15 atgaatagag atataaaaaa agaagttccaa ctaaatacag citcaaatgct agtaaaatgt 6 O ttagaa.gc.cg aaggagtaaa gta catctitt gg tatt cct gtgaagaaaa cct agaaata 12 O atgaatgcaa titt cagattic aactattgaa tittat cacaa ccc.gt catga gcaaggtgct 18O gcattt atgg ccgacgttta tigacgttta acaggaaaag Caggtgtttg cct at Calaca 24 O

Ctaggaccag gtgccact aa cittagtaact ggtgtag cag atgctgatag tatggtgct 3OO US 2016/0160223 A1 Jun. 9, 2016 35

- Continued ccggttgttg ct attacagg tdaagtaggit actgaaagaa toatataac atcgcaccala 360 tttittagacc tittgcaaaat gttcgaacca at cacaaaga gaagitaalaca aatcgttcgt. 42O cctgat actg taagtgagat tataagacitt gtttittaagt atgctgaaag tdaaaagcct 48O ggagcatgcc acattgattt acctgtaaat attgcaaaaa tdcc.cgtagg togctittagaa 54 O aagc ctittgg aaaagaagat tccaccalaag galacatgcag atttatcaac aattgaggaa 6OO gctgcaagtgaaatcttcaa agcaaaaaat cott attatct tagctggaag cqgtgctata 660 agaggaaatt cittcaaaagc tigttacggaa tittgcaacta aattgaaaat tccagtaatt 72 O aatacgatga tiggcaaaagg tattatt coa atggataa.ca agtatt caat gtgga caata 78O gg tatt coac aaaaagatta totaaataaa attattgaag aggctgattt agtaattaca 84 O attggatatg at attgtaga atatgcc.cca tocaaatgga atataaatgg gga cattaaa 9 OO attgtgcata t catgcaag accat cacac atcaataaac tittat cagoc catagtagaa 96.O gtagttggtg at attt caga tigctictatac aatatattga gaagaacttic tagcaaagat O2O galaccagtaa aagctittgga aattaaatca gaaatgctag ctgaacatga aagctatgca O8O aatgacaatig cittitt.ccaat gaaac cccaa agaattittaa atgatgttag aaagg to atg 14 O ggaccacatg acattgtcat at Cagatgta ggtgcc cata aaatgtggat tccagaCat 2OO tata actgct atgagcc.caa tacatgitatt atttcaaacg gttittgctac aatggg tatt 26 O ggtgttcCaggtgcaattgc agcCalaatta attaatcCag ataaaaaagt attggct att 32O gttggtgatg gcggtttcat gatgaataat Caagaattag aaa.ca.gc.cct acg tattaaa 38O actic caattig tagttittaat atttaatgac agtaactacg gtttaataaa gtggaaacaa 44 O gaagaacact atggtaaaag ctgttatgta gattt tacta atccagacitt totaaagctt SOO gCagaaagta t tatgcaaa aggat atcga gtagaaaaag Cagaagattit aattic caact 560 ttagaagaag ctittcaaaca aaatgtacct gcagttattg attgttcaagt tdactatggit 62O gaaaatataa agcttacaaa gcatttaaaa gaagtttatgaaaatatgta a 671

<210s, SEQ ID NO 16 &211s LENGTH: 556 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum LZ1561 <4 OOs, SEQUENCE: 16 Met Asn Arg Asp Ile Llys Lys Glu Val Glin Lieu. Asn. Thir Ala Gln Met 1. 5 1O 15 Lieu Val Lys Cys Lieu. Glu Ala Glu Gly Wall Lys Tyr Ile Phe Gly Ile 2O 25 3O Pro Gly Glu Glu Asn Lieu. Glu Ile Met Asn Ala Ile Ser Asp Ser Thr 35 4 O 45 Ile Glu Phe Ile Thr Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala SO 55 6 O

Asp Val Tyr Gly Arg Lieu. Thr Gly Lys Ala Gly Val Cys Lieu. Ser Thr 65 70 7s 8O

Lieu. Gly Pro Gly Ala Thr Asn Lieu Val Thr Gly Val Ala Asp Ala Asp 85 90 95

Ser Asp Gly Ala Pro Val Val Ala Ile Thr Gly Glin Val Gly Thr Glu 1OO 105 11 O US 2016/0160223 A1 Jun. 9, 2016 36

- Continued Arg Met His Ile Thr Ser His Glin Phe Lieu. Asp Lieu. Cys Llys Met Phe 115 12 O 125 Glu Pro Ile Thr Lys Arg Ser Lys Glin Ile Val Arg Pro Asp Thr Val 13 O 135 14 O Ser Glu Ile Ile Arg Lieu Val Phe Llys Tyr Ala Glu Ser Glu Lys Pro 145 150 155 160 Gly Ala Cys His Ile Asp Lieu Pro Val Asn. Ile Ala Lys Met Pro Val 1.65 17O 17s Gly Ala Lieu. Glu Lys Pro Lieu. Glu Lys Lys Ile Pro Pro Lys Glu. His 18O 185 19 O Ala Asp Lieu. Ser Thir Ile Glu Glu Ala Ala Ser Glu Ile Phe Lys Ala 195 2OO 2O5 Lys Asn Pro Ile Ile Lieu Ala Gly Ser Gly Ala Ile Arg Gly Asn. Ser 21 O 215 22O Ser Lys Ala Val Thr Glu Phe Ala Thir Lys Lieu Lys Ile Pro Val Ile 225 23 O 235 24 O Asn Thr Met Met Ala Lys Gly Ile Ile Pro Met Asp Asn Llys Tyr Ser 245 250 255 Met Trp Thir Ile Gly Ile Pro Gln Lys Asp Tyr Val Asn Lys Ile Ile 26 O 265 27 O Glu Glu Ala Asp Lieu Val Ile Thir Ile Gly Tyr Asp Ile Val Glu Tyr 27s 28O 285 Ala Pro Ser Llys Trp ASn Ile ASn Gly Asp Ile Lys Ile Val His Ile 29 O 295 3 OO Asp Ala Arg Pro Ser His Ile Asn Llys Lieu. Tyr Glin Pro Ile Val Glu 3. OS 310 315 32O Val Val Gly Asp Ile Ser Asp Ala Lieu. Tyr Asn. Ile Lieu. Arg Arg Thr 3.25 330 335 Ser Ser Lys Asp Glu Pro Wall Lys Ala Lieu. Glu Ile Llys Ser Glu Met 34 O 345 35. O Lieu Ala Glu. His Glu Ser Tyr Ala Asn Asp Asn Ala Phe Pro Met Lys 355 360 365 Pro Glin Arg Ile Lieu. Asn Asp Val Arg Llys Val Met Gly Pro His Asp 37 O 375 38O Ile Val Ile Ser Asp Val Gly Ala His Llys Met Trp Ile Ala Arg His 385 390 395 4 OO Tyr Asn Cys Tyr Glu Pro Asn Thr Cys Ile Ile Ser Asn Gly Phe Ala 4 OS 41O 415 Thir Met Gly Ile Gly Val Pro Gly Ala Ile Ala Ala Lys Lieu. Ile Asn 42O 425 43 O Pro Asp Llys Llys Val Lieu Ala Ile Val Gly Asp Gly Gly Phe Met Met 435 44 O 445

Asn Asn Glin Glu Lieu. Glu Thir Ala Lieu. Arg Ile Llys Thr Pro Ile Val 450 45.5 460

Val Lieu. Ile Phe Asn Asp Ser Asn Tyr Gly Lieu. Ile Llys Trp Llys Glin 465 470 47s 48O Glu Glu. His Tyr Gly Lys Ser Cys Tyr Val Asp Phe Thr Asn Pro Asp 485 490 495

Phe Val Llys Lieu Ala Glu Ser Met Tyr Ala Lys Gly Tyr Arg Val Glu SOO 505 51O

Lys Ala Glu Asp Lieu. Ile Pro Thir Lieu. Glu Glu Ala Phe Lys Glin Asn US 2016/0160223 A1 Jun. 9, 2016 37

- Continued

515 52O 525 Val Pro Ala Val Ile Asp Cys Glin Val Asp Tyr Gly Glu Asn. Ile Llys 53 O 535 54 O Lieu. Thir Lys His Lieu Lys Glu Val Tyr Glu Asn Met 5.45 550 555

<210s, SEQ ID NO 17 &211s LENGTH: 168O &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized Clostridium autoethanogenum LZ1561. AlsS acetolactate synthase

<4 OOs, SEQUENCE: 17 catatgaata gagatataaa aaaagaagta caattaaata citgcacaaat gttagtaaaa 6 O tgtttagaag Cagaaggagt aaaatatata tittggaatac Ctggagaaga aaatttagaa 12 O ataatgaatg caat atctga tag tactata gaatttataa citact agaca tdaacaagga 18O gcagcattta tigcagatgt atatggaaga ttaactggaa aagctggagt ttgtttalagt 24 O actittaggac Ctggagcaac taatttagta actggagtag cagatgcaga tagtgatgga 3OO gCacct gtag tag caataac tigacaagta ggaactgaaa gaatgcatat alactagt cat 360 caattitt tag atttatgtaa aatgtttgaa cctataacta aaagaagitaa acaaatagta 42O agacCtgata Ctgtaagtga aataataaga t tagt attta aatatgcaga aagtgaaaaa 480 cctggagcat gtcatataga tttacctgta aatatagcaa aaatgcctgt toggagcatta 54 O gaaaaacctt tagaaaaaaa aatacctic ct aaagaacatg cagatttaag tacaatagaa 6OO gaag cagcat Ctgaaatatt taaagcaaaa aatcc tataa tattagctgg aagtggagca 660 ataagaggaa at agtagtaa agcagtaact gaatttgcaa citaaattaaa aatacct gta 72 O ataaatacta tdatggcaaa aggaataata cctatggata ataaatatag tatgtgg act 78O ataggaatac citcaaaaaga t tatgtaaat aaaataatag aagaa.gctga tittagtaata 84 O actataggat atgatatagt agaatatgca cctagtaaat ggaatataaa toggagatata 9 OO aaaatagtac atatagatgc aag acctagt catataaata aattatat ca acctatagta 96.O gaagtagttg gagatataag togatgcatta tataatatat taagaagaac tagttcaaaa O2O gatgaacctg taaaag catt agaaataaaa agtgaaatgt tag cagaac a tigaaagttat O8O gcaaatgata atgcatttico tatgaaac ct caaagaatat taaatgatgt aagaaaagta 14 O atgggacctic atgatatagt aataagtgat gttggagcac ataaaatgtg gatagcaaga 2OO cattataatt gttatgaacc taatacttgt ataataagta atggatttgc aacaatggga 26 O ataggagtac ctdgagcaat agcagcaaaa ttaataaatc ctdataaaaa agt attagca 32O at agttggag atggaggatt tatgatgaat aat caagaat tagaaactgc attaagaata 38O aaaact cota tagtag tatt aatatttaat gatagtaatt atggattaat aaaatggaaa 44 O caagaagaac attatggaaa aagttgttat gtagattitta ctaatcc toga titttgtaaaa SOO ttagcaga aa gtatgtatgc aaaaggat at agagtagaala aag cagaaga tittaatacct 560 actittagaag aag catttaa acaaaatgta cct gcagtaa tagattgtca agtagattat 62O ggagaaaata taaaattaac taalacattta aaagaagitat atgaaaatat gtaagagctic 68O US 2016/0160223 A1 Jun. 9, 2016 38

- Continued

<210s, SEQ ID NO 18 &211s LENGTH: 556 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized Clostridium autoethanogenum LZ1561. AlsS acetolactate synthase

<4 OOs, SEQUENCE: 18 Met Asn Arg Asp Ile Llys Lys Glu Val Glin Lieu. Asn. Thir Ala Gln Met 1. 5 1O 15 Lieu Val Lys Cys Lieu. Glu Ala Glu Gly Wall Lys Tyr Ile Phe Gly Ile 2O 25 3O Pro Gly Glu Glu Asn Lieu. Glu Ile Met Asn Ala Ile Ser Asp Ser Thr 35 4 O 45 Ile Glu Phe Ile Thr Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala SO 55 6 O Asp Val Tyr Gly Arg Lieu. Thr Gly Lys Ala Gly Val Cys Lieu. Ser Thr 65 70 7s 8O Lieu. Gly Pro Gly Ala Thr Asn Lieu Val Thr Gly Val Ala Asp Ala Asp 85 90 95 Ser Asp Gly Ala Pro Val Val Ala Ile Thr Gly Glin Val Gly Thr Glu 1OO 105 11 O Arg Met His Ile Thr Ser His Glin Phe Lieu. Asp Lieu. Cys Llys Met Phe 115 12 O 125 Glu Pro Ile Thr Lys Arg Ser Lys Glin Ile Val Arg Pro Asp Thr Val 13 O 135 14 O Ser Glu Ile Ile Arg Lieu Val Phe Llys Tyr Ala Glu Ser Glu Lys Pro 145 150 155 160 Gly Ala Cys His Ile Asp Lieu Pro Val Asn. Ile Ala Lys Met Pro Val 1.65 17O 17s Gly Ala Lieu. Glu Lys Pro Lieu. Glu Lys Lys Ile Pro Pro Lys Glu. His 18O 185 19 O Ala Asp Lieu. Ser Thir Ile Glu Glu Ala Ala Ser Glu Ile Phe Lys Ala 195 2OO 2O5 Lys Asn Pro Ile Ile Lieu Ala Gly Ser Gly Ala Ile Arg Gly Asn. Ser 21 O 215 22O Ser Lys Ala Val Thr Glu Phe Ala Thir Lys Lieu Lys Ile Pro Val Ile 225 23 O 235 24 O Asn Thr Met Met Ala Lys Gly Ile Ile Pro Met Asp Asn Llys Tyr Ser 245 250 255 Met Trp Thir Ile Gly Ile Pro Gln Lys Asp Tyr Val Asn Lys Ile Ile 26 O 265 27 O Glu Glu Ala Asp Lieu Val Ile Thir Ile Gly Tyr Asp Ile Val Glu Tyr 27s 28O 285 Ala Pro Ser Lys Trp Asn. Ile Asin Gly Asp Ile Llys Ile Val His Ile 29 O 295 3 OO Asp Ala Arg Pro Ser His Ile Asn Llys Lieu. Tyr Glin Pro Ile Val Glu 3. OS 310 315 32O

Val Val Gly Asp Ile Ser Asp Ala Lieu. Tyr Asn. Ile Lieu. Arg Arg Thr 3.25 330 335

Ser Ser Lys Asp Glu Pro Wall Lys Ala Lieu. Glu Ile Llys Ser Glu Met 34 O 345 35. O US 2016/0160223 A1 Jun. 9, 2016 39

- Continued Lieu Ala Glu. His Glu Ser Tyr Ala Asn Asp Asn Ala Phe Pro Met Lys 355 360 365 Pro Glin Arg Ile Lieu. Asn Asp Val Arg Llys Val Met Gly Pro His Asp 37 O 375 38O Ile Val Ile Ser Asp Val Gly Ala His Llys Met Trp Ile Ala Arg His 385 390 395 4 OO Tyr Asn Cys Tyr Glu Pro Asn Thr Cys Ile Ile Ser Asn Gly Phe Ala 4 OS 41O 415 Thir Met Gly Ile Gly Val Pro Gly Ala Ile Ala Ala Lys Lieu. Ile Asn 42O 425 43 O Pro Asp Llys Llys Val Lieu Ala Ile Val Gly Asp Gly Gly Phe Met Met 435 44 O 445 Asn Asn Glin Glu Lieu. Glu Thir Ala Lieu. Arg Ile Llys Thr Pro Ile Val 450 45.5 460 Val Lieu. Ile Phe Asn Asp Ser Asn Tyr Gly Lieu. Ile Llys Trp Llys Glin 465 470 47s 48O Glu Glu. His Tyr Gly Lys Ser Cys Tyr Val Asp Phe Thr Asn Pro Asp 485 490 495 Phe Val Llys Lieu Ala Glu Ser Met Tyr Ala Lys Gly Tyr Arg Val Glu SOO 505 51O Lys Ala Glu Asp Lieu. Ile Pro Thir Lieu. Glu Glu Ala Phe Lys Glin Asn 515 52O 525 Val Pro Ala Val Ile Asp Cys Glin Val Asp Tyr Gly Glu ASn Ile Llys 53 O 535 54 O Lieu. Thir Lys His Lieu Lys Glu Val Tyr Glu Asn Met 5.45 550 555

<210s, SEQ ID NO 19 &211s LENGTH: 1722 &212s. TYPE: DNA <213> ORGANISM; Bacillus subtilis

<4 OOs, SEQUENCE: 19 catatgacta aagcaactaa agaacaaaaa agtttagtaa aaaatagagg togcagaatta 6 O gtag tagatt gtttagtaga acaaggtgta act catgitat ttggaat acc tigtgcaaaa 12 O atagatgcag tatttgatgc attacaagat aaaggacctgaaataatagt agcaaga cat 18O gaacaaaatg cagcattt at ggcacaagca gtaggaagat taactggaala acctggtgta 24 O gtacttgtaa citagtggacc toggtgcatca aatttagcaa citggattatt aactgcaaat 3OO actgaaggtg atcCtgtagt agcattagct ggaaatgitaa talaga.gcaga tagattaaaa 360 agaact catc aaagtttaga taatgcagca ttatttcaac ctataactaa at attctgta 42O gaagtacaag atgtaaaaaa tatacctgaa goagtaacta atgcatttag aatagcaagt 48O gCaggacaag Ctggtgcagc titttgtaagt titt CCtcagg atgtag taala talagta act 54 O aatact aaaa atgitalaga.gc agtag cagca Cctaaattag gacctgcagc agatgatgca 6OO ataagtgcag caatagdaaa aatacaaact gcaaaattac ctdtag tatt agtaggaatg 660 aaaggtggta gacctgaagc aataaaag.ca gtaagaaaat tacttaaaaa agtacaatta 72 O c ctitttgtag aaactitatica agcagctgga actittaagta gagatttaga agat caatat 78O tittggaagaa taggattatt tagaaatcaa cct ggtgatt tattacttga acaag cagat 84 O gtag tattaa citataggata tdatccaata gaatatgatc ctaaattittg gaatataaat 9 OO US 2016/0160223 A1 Jun. 9, 2016 40

- Continued ggtgatagaa citataataca tttagatgaaataatagoag atatagat.ca togcatat caa 96.O cctgatttag aattaattgg agatatacct agtactataa at cacataga acatgatgca O2O gtaaaagtag aatttgcaga aagagaacaa aaaat actta gtgatttaaa acaatatatg O8O

Catgaaggtgaacaagtacc ticagattgg aaaagtgata gag cacatcc tittagaaata 14 O gtaaaagaat taagaaatgc agtagatgat catgtaactg. taacttgttga tataggat.ct 2OO catgcaat at gigatgagtag at attittaga agittatgaac ctitta actitt aatgataagt 26 O aatggaatgc aaacticttgg agtag catta Ccttgggcaa ttggagcatc tittagtaaaa 32O Cctggtgaaa aagtag taag titaagtggit gatggtggat ttcttitt tag tecaatggaa 38O ttagaaactg cagtaagatt aaaag cacct at agtacata tag tatggaa tdatagtact 44 O tatgatatgg tag catttica acaattaaaa aaatataata gaact agtgc agtagattitt SOO ggaalatatag atatagtaaa atatgcagaa agttittggag caa.caggatt aagagtagaa 560 agtic ctgatc aattagcaga tigt acttaga Cagggaatga atgcaga agg accagtaata 62O attgatgtac ctdtagatta tagtgataat ataaatttag caagtgataa attacctaaa 68O gaatttggag aattaatgaa alactaaag.ca ttataagagc tic 722

<210s, SEQ ID NO 2 O &211s LENGTH: 570 212. TYPE: PRT <213> ORGANISM; Bacillus subtilis

<4 OOs, SEQUENCE: 2O Met Thir Lys Ala Thr Lys Glu Glin Llys Ser Lieu Val Lys Asn Arg Gly 1. 5 1O 15 Ala Glu Lieu Val Val Asp Cys Lieu Val Glu Glin Gly Val Thr His Val 2O 25 3O Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Lieu. Glin 35 4 O 45 Asp Llys Gly Pro Glu Ile Ile Val Ala Arg His Glu Glin Asn Ala Ala SO 55 6 O Phe Met Ala Glin Ala Val Gly Arg Lieu. Thr Gly Llys Pro Gly Val Val 65 70 7s 8O Lieu Val Thir Ser Gly Pro Gly Ala Ser Asn Lieu Ala Thr Gly Lieu. Lieu. 85 90 95 Thir Ala Asn Thr Glu Gly Asp Pro Val Val Ala Lieu Ala Gly Asn. Wall 1OO 105 11 O Ile Arg Ala Asp Arg Lieu Lys Arg Thr His Glin Ser Lieu. Asp Asn Ala 115 12 O 125 Ala Leu Phe Glin Pro Ile Thr Lys Tyr Ser Val Glu Val Glin Asp Val 13 O 135 14 O

Lys Asn. Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser Ala 145 150 155 160

Gly Glin Ala Gly Ala Ala Phe Val Ser Phe Pro Glin Asp Val Val Asn 1.65 17O 17s

Glu Val Thir Asn. Thir Lys Asn Val Arg Ala Val Ala Ala Pro Llys Lieu. 18O 185 19 O

Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile Glin 195 2OO 2O5 US 2016/0160223 A1 Jun. 9, 2016 41

- Continued Thir Ala Lys Lieu Pro Val Val Lieu Val Gly Met Lys Gly Gly Arg Pro 21 O 215 22O Glu Ala Ile Lys Ala Val Arg Llys Lieu. Lieu Lys Llys Val Glin Lieu Pro 225 23 O 235 24 O Phe Val Glu Thir Tyr Glin Ala Ala Gly. Thir Lieu. Ser Arg Asp Lieu. Glu 245 250 255 Asp Glin Tyr Phe Gly Arg Ile Gly Lieu. Phe Arg Asn Gln Pro Gly Asp 26 O 265 27 O Lieu. Lieu. Lieu. Glu Glin Ala Asp Val Val Lieu. Thir Ile Gly Tyr Asp Pro 27s 28O 285 Ile Glu Tyr Asp Pro Llys Phe Trp Asn. Ile Asn Gly Asp Arg Thir Ile 29 O 295 3 OO Ile His Lieu. Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Glin Pro 3. OS 310 315 32O Asp Lieu. Glu Lieu. Ile Gly Asp Ile Pro Ser Thir Ile Asn His Ile Glu 3.25 330 335 His Asp Ala Val Llys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile Lieu. 34 O 345 35. O Ser Asp Lieu Lys Glin Tyr Met His Glu Gly Glu Glin Val Pro Ala Asp 355 360 365 Trp Llys Ser Asp Arg Ala His Pro Lieu. Glu Ile Val Lys Glu Lieu. Arg 37 O 375 38O Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 4 OO Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu. Thir Lieu. 4 OS 41O 415 Met Ile Ser Asn Gly Met Gln Thr Lieu. Gly Val Ala Leu Pro Trp Ala 42O 425 43 O Ile Gly Ala Ser Lieu Val Llys Pro Gly Glu Lys Val Val Ser Val Ser 435 44 O 445 Gly Asp Gly Gly Phe Lieu. Phe Ser Ala Met Glu Lieu. Glu Thir Ala Val 450 45.5 460 Arg Lieu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr Tyr 465 470 47s 48O Asp Met Val Ala Phe Glin Glin Lieu Lys Llys Tyr Asn Arg Thir Ser Ala 485 490 495 Val Asp Phe Gly Asn. Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe Gly SOO 505 51O Ala Thr Gly Lieu. Arg Val Glu Ser Pro Asp Glin Lieu Ala Asp Val Lieu 515 52O 525 Arg Glin Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro Val 53 O 535 54 O

Asp Tyr Ser Asp Asn. Ile Asn Lieu Ala Ser Asp Llys Lieu Pro Lys Glu 5.45 550 555 560

Phe Gly Glu Lieu Met Lys Thr Lys Ala Lieu. 565 st O

<210s, SEQ ID NO 21 &211s LENGTH: 720 &212s. TYPE: DNA <213> ORGANISM: Clostridium autoethanogenum LZ1561

<4 OOs, SEQUENCE: 21 US 2016/0160223 A1 Jun. 9, 2016 42

- Continued atggatgatg aggtgaaagt cccaaaccat atatat caaa tdtctacaat aaatgcactt 6 O gttt cqgggc tig tatgatgg ctgtgttt ca ttatctaaac ttcttaaaaa aggaaactitt 12 O ggtataggta cittittaaagg totagatggit gaactaactic ttittaaatgg aactttittat 18O aggactaaac ctdatggcag cqtatacgta tdttccaaaa acgitatic cq t t ccttittgct 24 O gtag to actgaactggaaaa ttataatact tataatatt c aaaatcgtac ttct tatgaa 3OO gatataagaa aagaattgga cagctittata gaaagcaaaa atatattitta togctttctat 360 atggaaggta aatttaatta totaaaaa.ca cqtactgttg taaaacagaa tatgc ctitat 42O aagcct atgg ctgaagttgt taaagat cag cctatgtttgaatataacgg tttgatgga 48O tatgtggttggatttaggtg tcc tatt at gttgaaggcc ttaatgtc.cc tigatat cat 54 O titt cattt ca taaataaaga taagaaattt ggtggacata taagtgaatt titc cattgaa 6OO aatgcgaagg tittatgtaca gaactgttct togctittagga tiggaact tcc taaaaatgaa 660 agtttittata atatggaagt acaagataga aacgatgaga taacaagtgt taaaaataa 72 O

<210s, SEQ ID NO 22 &211s LENGTH: 239 212. TYPE: PRT <213> ORGANISM: Clostridium autoethanogenum LZ1561

<4 OOs, SEQUENCE: 22 Met Asp Asp Glu Val Llys Val Pro Asn His Ile Tyr Gln Met Ser Thr 1. 5 1O 15 Ile Asn Ala Lieu Val Ser Gly Lieu. Tyr Asp Gly Cys Val Ser Lieu. Ser 2O 25 3O Llys Lieu. Lieu Lys Lys Gly Asn. Phe Gly Ile Gly Thr Phe Lys Gly Lieu. 35 4 O 45 Asp Gly Glu Lieu. Thir Lieu. Lieu. Asn Gly Thr Phe Tyr Arg Thr Llys Pro SO 55 6 O Asp Gly Ser Val Tyr Val Cys Ser Lys Asn Val Ser Val Pro Phe Ala 65 70 7s 8O Val Val Thr Glu Lieu. Glu Asn Tyr Asn. Thir Tyr Asn. Ile Glin Asn Arg 85 90 95 Thir Ser Tyr Glu Asp Ile Arg Lys Glu Lieu. Asp Ser Phe Ile Glu Ser 1OO 105 11 O Lys Asn Ile Phe Tyr Ala Phe Tyr Met Glu Gly Llys Phe Asn Tyr Val 115 12 O 125 Lys Thr Arg Thr Val Val Lys Glin Asn Met Pro Tyr Lys Pro Met Ala 13 O 135 14 O Glu Val Val Lys Asp Gln Pro Met Phe Glu Tyr Asn Gly Val Asp Gly 145 150 155 160

Tyr Val Val Gly Phe Arg Cys Pro Asp Tyr Val Glu Gly Lieu. Asn Val 1.65 17O 17s Pro Gly Tyr His Phe His Phe Ile Asn Lys Asp Llys Llys Phe Gly Gly 18O 185 19 O

His Ile Ser Glu Phe Ser Ile Glu Asn Ala Lys Val Tyr Val Glin Asn 195 2OO 2O5

Cys Ser Cys Phe Arg Met Glu Lieu Pro Lys Asn Glu Ser Phe Tyr Asn 21 O 215 22O

Met Glu Val Glin Asp Arg Asn Asp Glu Ile Thir Ser Val Glu Lys US 2016/0160223 A1 Jun. 9, 2016 43

- Continued

225 23 O 235

<210s, SEQ ID NO 23 &211s LENGTH: 745 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized Clostridium autoethanogenum LZ1561 acetolactate decarboxylase

<4 OOs, SEQUENCE: 23 gagotcagga ggtaactaaa tigatgatga agtaaaagta Cctaatcat a tatat calaat 6 O gagtactata aatgcattag taagtggatt atatgatgga tigtgtaagtt tat ctaaatt 12 O attaaaaaaa goaaattittg gaataggaac ttittaaagga ttagatggag aatta actitt 18O attaaatgga actttittata gaactaaacc tdatggaagt gtatatgtat gtagtaaaaa 24 O tgtaagtgta ccttittgcag tagtaactga attagaaaat tataatactt ataatataca 3OO aaatagaact tct tatgaag atataagaaa agaattagat agttittatagaaagtaaaaa 360 tatattittat gcattittata toggaaggaaa atttaattat gtaaaaact a gaactgtagt 42O aaaacaaaat atgccttata aacctatgc agaagtagta aaagat caac ctatotttga 48O atataatgga gtagatggat atgtag tagg atttagatgt cct gattatg tagaaggatt 54 O aaatgtacct ggatat catt tt catttitat aaataaagat aaaaaatttg gagga catat 6OO aagtgaattt agtatagaaa atgcaaaagt atatgtacaa aattgtagtt gttittagaat 660 ggaattacct aaaaatgaaa gtttittataa tatggaagta caagatagaa atgatgaaat 72 O aact agtgta gaaaaataag gtacc 74.

<210s, SEQ ID NO 24 &211s LENGTH: 239 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Codon-optimized Clostridium autoethanogenum LZ1561 acetolactate decarboxylase

<4 OOs, SEQUENCE: 24 Met Asp Asp Glu Val Lys Val Pro Asn His Ile Tyr Gln Met Ser Thr 1. 5 1O 15 Ile Asn Ala Lieu Val Ser Gly Lieu. Tyr Asp Gly Cys Val Ser Lieu. Ser 2O 25 3O Llys Lieu. Lieu Lys Lys Gly Asn. Phe Gly Ile Gly Thr Phe Lys Gly Lieu. 35 4 O 45 Asp Gly Glu Lieu. Thir Lieu. Lieu. Asn Gly Thr Phe Tyr Arg Thr Llys Pro SO 55 6 O

Asp Gly Ser Val Tyr Val Cys Ser Lys Asn Val Ser Val Pro Phe Ala 65 70 7s 8O

Val Val Thr Glu Lieu. Glu Asn Tyr Asn. Thir Tyr Asn. Ile Glin Asn Arg 85 90 95

Thir Ser Tyr Glu Asp Ile Arg Lys Glu Lieu. Asp Ser Phe Ile Glu Ser 1OO 105 11 O

Lys Asn Ile Phe Tyr Ala Phe Tyr Met Glu Gly Llys Phe Asn Tyr Val 115 12 O 125

Lys Thr Arg Thr Val Val Lys Glin Asn Met Pro Tyr Lys Pro Met Ala 13 O 135 14 O US 2016/0160223 A1 Jun. 9, 2016 44

- Continued

Glu Val Val Lys Asp Gln Pro Met Phe Glu Tyr Asn Gly Val Asp Gly 145 150 155 160 Tyr Val Val Gly Phe Arg Cys Pro Asp Tyr Val Glu Gly Lieu. Asn Val 1.65 17O 17s Pro Gly Tyr His Phe His Phe Ile Asn Lys Asp Llys Llys Phe Gly Gly 18O 185 19 O His Ile Ser Glu Phe Ser Ile Glu Asn Ala Lys Val Tyr Val Glin Asn 195 2OO 2O5 Cys Ser Cys Phe Arg Met Glu Lieu Pro Lys Asn Glu Ser Phe Tyr Asn 21 O 215 22O Met Glu Val Glin Asp Arg Asn Asp Glu Ile Thir Ser Val Glu Lys 225 23 O 235

<210s, SEQ ID NO 25 &211s LENGTH: 806 &212s. TYPE: DNA <213> ORGANISM: Aeromonas hydrophila <4 OOs, SEQUENCE: 25 gagotctaag gaggtoggac atggaaacta at agtagttg tattgtgca atagaaataa 6 O gtcaacaatt to aagatgg Caggcaagac alaggtggtgg talagtatat caaagtagt c 12 O titatgagtgc attattagct ggtgtatatgaaggtgaaac tactatgca gatt tactta 18O gaCatggtga ttttggatta galacttitta atagattaga tiggtgaatta at agcatttg 24 O aaagacaaat a catcaatta aaa.gcagatg gaagtgcaag acctgcaaga gcagaacaaa 3OO aaactic ctitt togcagtaatg act catttta gaccttgttt acaaagaaga tittgcacatc 360 Ctttalagtag agaagaaata cat cagtggg tagatagatt agtaggaact gataatgitat 42O ttgtag catt tag acttgat ggattatttgaacaa.gcaca agtaagaact gtaccttgtc 48O aaagtic ct cottataa acct atgttagaag caatagaagc acaac ctitta tittagttitta 54 O gtttalagaag aggaactitta gtaggattta gatgtcCtcc titttgtacag ggaataaatg 6OO tagcaggata t catgaac at tittataactgaagatagaag aggtggtgga catat attag 660 attatgcaat giggacatgga caattacaat taagtgtagt acaa.cat citt aatatagaat 72 O tacctagaaa toctdcattt caacaagcag at cittaatcc togcagattta gatagagcaa. 78O talaga.gcagc agaaggataa ggt acc 806

<210s, SEQ ID NO 26 &211s LENGTH: 259 212. TYPE: PRT <213> ORGANISM: Aeromonas hydrophila <4 OOs, SEQUENCE: 26 Met Glu Thir Asn Ser Ser Cys Asp Cys Ala Ile Glu Ile Ser Glin Glin 1. 5 1O 15

Phe Ala Arg Trp Glin Ala Arg Glin Gly Gly Gly Glu Val Tyr Glin Ser 2O 25 3O

Ser Leu Met Ser Ala Lieu. Leu Ala Gly Val Tyr Glu Gly Glu. Thir Thr 35 4 O 45 Met Ala Asp Lieu. Lieu. Arg His Gly Asp Phe Gly Lieu. Gly Thr Phe Asn SO 55 6 O

Arg Lieu. Asp Gly Glu Lieu. Ile Ala Phe Glu Arg Glin Ile His Glin Lieu US 2016/0160223 A1 Jun. 9, 2016 45

- Continued

Lys Ala Asp Gly Ser Ala Arg Pro Ala Arg Ala Glu Gln Lys Thr Pro 85 90 95 Phe Ala Val Met Thr His Phe Arg Pro Cys Lieu. Glin Arg Arg Phe Ala 1OO 105 11 O His Pro Lieu. Ser Arg Glu Glu Ile His Glin Trp Val Asp Arg Lieu Val 115 12 O 125 Gly. Thir Asp Asin Val Phe Val Ala Phe Arg Lieu. Asp Gly Lieu. Phe Glu 13 O 135 14 O Glin Ala Glin Val Arg Thr Val Pro Cys Glin Ser Pro Pro Tyr Llys Pro 145 150 155 160 Met Lieu. Glu Ala Ile Glu Ala Glin Pro Lieu. Phe Ser Phe Ser Lieu. Arg 1.65 17O 17s Arg Gly Thr Lieu Val Gly Phe Arg Cys Pro Pro Phe Val Glin Gly Ile 18O 185 19 O Asn Val Ala Gly Tyr His Glu. His Phe Ile Thr Glu Asp Arg Arg Gly 195 2OO 2O5 Gly Gly. His Ile Lieu. Asp Tyr Ala Met Gly His Gly Glin Lieu. Glin Lieu. 21 O 215 22O Ser Val Val Glin His Lieu. Asn. Ile Glu Lieu Pro Arg ASn Pro Ala Phe 225 23 O 235 24 O Glin Glin Ala Asp Lieu. Asn Pro Ala Asp Lieu. Asp Arg Ala Ile Arg Ala 245 250 255 Ala Glu Gly

<210s, SEQ ID NO 27 &211s LENGTH: 761 &212s. TYPE: DNA <213> ORGANISM: Leuconostoc lactis

<4 OOs, SEQUENCE: 27 gagctictaag gaggtoggac atgact catc aatataataa aatgagtaga ttatat caac 6 O atggaactitt agcaatgtta atgggaaaac aaatgcaggg alactata act gtagcagaac 12 O ttagacaa.ca tdtgatact ggaataggaa citctt actgg attagatggit galagtaatac 18O ttcttgatgg togaagttitat caa.gcacaga gtgatggaca agtaaat cat ataactaatc 24 O citgatactitt aatgcc ttitt gcaagtgtac attittgatgc acc tact cag cagttacctt 3OO ttagt caggt agattittagt actittaagtg caaaacttaa agcagaacaa ttacaaaatg 360 tatttgcago agtgaaattt catggtgaat ttagtagagt acatgtaaga atagotccta 42O aacaagtacct cottatcct t cattacttg cagtagcaga aaatcaacct gaatttgaaa 48O gagaac acgt aactggaact gtag taggat actittgcacc ticaaatattt aatgg accta 54 O ctgcagcagg atggcatgta catttitctta gtgatgatct t caatttgca gga catatat 6OO tagattittag togcaagttcaa ataagtggaa citttacaaat atttgatgaa tttgtacaac 660 atttacct at acatgatcct gcatatagag aaatgactitt agattittgat ggatt attag 72 O

Ctggaataga agcaagtgaa ggtggacaac aataagg tac C 761

<210s, SEQ ID NO 28 &211s LENGTH: 244 212. TYPE: PRT <213> ORGANISM: Leuconostoc lactis US 2016/0160223 A1 Jun. 9, 2016 46

- Continued

<4 OOs, SEQUENCE: 28 Met Thr His Glin Tyr Asn Met Ser Arg Lieu. Tyr Glin His Gly Thr 1. 15

Lell Ala Met Luell Met Gly Gln Met Glin Gly Thir Ile Thir Wall Ala 25

Glu Lieu. Arg Glin His Gly Asp Thir Gly Ile Gly Thir Lieu. Thir Gly Lieu. 35 4 O 45

Asp Gly Glu Wall Ile Lell Lell Asp Gly Glu Wall Tyr Glin Ala Glin Ser SO 55 6 O

Asp Gly Glin Wall Asn His Ile Thir ASn Pro Asp Thir Lieu. Met Pro Phe 65 70 7s 8O

Ala Ser Wall His Phe Asp Ala Pro Thr Gin Glin Leu Pro Phe Ser Glin 85 90 95

Wall Asp Phe Ser Thir Lieu. Ser Ala Lys Luell Lys Ala Glu Glin Lieu. Glin 105 11 O

Asn Wall Phe Ala Ala Wall Phe His Gly Glu Phe Ser Arg Wall His 115 12 O 125

Wall Arg Ile Ala Pro Glin Wall Pro Pro Pro Ser Luell Lieu Ala 13 O 135 14 O

Wall Ala Glu Asn Glin Pro Glu Phe Glu Arg Glu His Wall Thir Gly Thir 145 150 155 160

Val Val Gly Tyr Phe Ala Pro Glin Ile Phe ASn Gly Pro Thr Ala Ala 1.65 17O 17s

Gly Trp His Wall His Phe Lieu. Ser Asp Asp Leul Glin Phe Ala Gly His 18O 185 19 O

Ile Lieu. Asp Phe Ser Ala Ser Glin Ile Ser Gly Thir Lieu. Glin Ile Phe 195

Asp Glu Phe Wall Glin His Leul Pro Ile His Asp Pro Ala Arg Glu 21 O 215

Met Thir Lieu. Asp Phe Asp Gly Lieu. Lieu Ala Gly Ile Glu Ala Ser Glu 225 23 O 235 24 O Gly Gly Glin Glin

1. A recombinant, carboxydotrophic Clostridium bacte 8. The bacterium of claim 1, wherein the enzyme is over rium comprising one or more enzymes selected from the expressed endogenous IlvB ORF2059 acetolactate synthase, group consisting of pyruvate:ferredoxin oxidoreductase (EC overexpressed endogenous IlvB ORF2336 acetolactate syn 1.2.7.1), acetolactate synthase (EC 2.2.1.6), and acetolactate thase, overexpressed endogenous IlvN acetolactate synthase, decarboxylase (EC 4.1.1.5), wherein each enzyme is an over or overexpressed endogenous Alss acetolactate synthase. expressed endogenous enzyme, a mutated endogenous 9. The bacterium of claim 1, wherein the enzyme is enzyme, or an exogenous enzyme. mutated endogenous IlvN acetolactate synthase. 2. The bacterium of claim 1, comprising pyruvate:ferre 10. The bacterium of claim 1, wherein the enzyme is exog doxin oxidoreductase and acetolactate synthase. enous Bacillus subtilis acetolactate synthase. 3. The bacterium of claim 1, comprising pyruvate:ferre 11. The bacterium of claim 1, wherein the enzyme is over doxin oxidoreductase and acetolactate decarboxylase. expressed endogenous AlsD acetolactate decarboxylase or 4. The bacterium of claim 1, comprising acetolactate syn thase and acetolactate decarboxylase. overexpressed endogenous BudA acetolactate decarboxy 5. The bacterium of claim 1, comprising pyruvate:ferre lase. doxin oxidoreductase, acetolactate synthase, and acetolactate 12. The bacterium of claim 1, wherein the enzyme is exog decarboxylase. enous Aeromonas hydrophila acetolactate decarboxylase. 6. The bacterium of claim 1, wherein the enzyme is over 13. The bacterium of claim 1, wherein the enzyme is exog expressed endogenous pyruvate:ferredoxin oxidoreductase. enous Leuconostoc lactis acetolactate decarboxylase. 7. The bacterium of claim 1, wherein the enzyme is exog 14. The bacterium of claim 1, wherein the bacterium is enous Desulfovibrio africanus pyruvate:ferredoxin oxi derived from Clostridium autoethanogenum, Clostridium doreductase. liungdahli, or Clostridium ragsdalei. US 2016/0160223 A1 Jun. 9, 2016 47

15. The bacterium of claim 1, wherein the bacterium is derived from Clostridium autoethanogenium. 16. The bacterium of claim 1, wherein the bacterium is derived from Clostridium autoethanogenium deposited under DSMZ Accession No. DSM23693. 17. A method of producing a fermentation product, com prising fermenting the bacterium of claim 1 in the presence of a gaseous Substrate comprising CO to produce a fermentation product. 18. The method of claim 17, wherein the fermentation product is selected from the group consisting of ethanol, butanol, isopropanol, isobutanol, higher alcohols, butanediol. 2,3-butanediol. Succinate, isoprenoids, fatty acids, biopoly mers, and mixtures thereof. 19. The method of claim 18, wherein the fermentation product is 2,3-butanediol. k k k k k