MICROORGANISMS FOR PRODUCING SUCCINATE AND METHODS RELATED

THERETO

[001] This application claims the benefit of priority of United States Provisional application serial No. 61/502,838, filed June 29, 2011, the entire contents of which are incorporated herein by reference.

[002] Incorporated herein by reference is the Sequence Listing being concurrently submitted via EFS-Web as an ASCII text file named 12956-153-228_SEQLIST.TXT, created June 27, 2012, and being 77,796 bytes in size.

BACKGROUND OF THE INVENTION

[003] The present invention relates generally to design and engineering of organisms, more particularly to organisms having increased succinate biosynthesis capability.

[004] Succinate is a compound of commercial interest due to its use as a precursor to commodity chemicals in the food, pharmaceutical, detergent and polymer industries.

Biological succinate production is also a green process where the greenhouse gas C0 ₂ must be fixed into succinate during sugar fermentation.

[005] Despite efforts to develop bacterial strains producing increased succinate yields, many approaches previously employed have several drawbacks which hinder applicability in commercial settings. For example, many such strains generally are unstable in commercial fermentation processes due to selective pressures favoring the unaltered or wild-type parental counterparts.

[006] Thus, there exists a need for microorganisms having commercially beneficial characteristics of increased production of succinate. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

[007] The invention provides microbial organisms comprising a genetic modification, including one or more genetic modifications, conferring to the microbial organism an increased production of succinate relative to the microbial organism in the absence of the genetic modification. The microbial organism additionally comprises pathways for reductive TCA and/or CO dehydrogenase and/or H ₂ hydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[008] Figure 1 shows exemplary pathways for the production of succinate from glucose. Oxidative TCA cycle enzymes and enzymes for the conversion of phosphoenolpyruvate to acetyl-CoA are required to produce succinate at the maximum theoretical yield (1.71 mol succinate/mol glucose). Note that several other carbohydrates can be converted to

phosphoenolpyruvate and succinate including xylose, arabinose, galactose, and glycerol.

[009] Figure 2 shows exemplary pathways for fixation of C0 ₂ to succinate using the reductive TCA cycle, various anapleurotic reactions, and enzymes for the extraction of reducing equivalents from CO and H ₂ .

[010] Figure 3 shows typical pathways for the production of succinate from glucose, C0 ₂ or CO, and reducing equivalents (for example, CO or H ₂ ) at a theoretical yield of 2.0 mol succinate/mol glucose. Oxidative TCA cycle enzymes and enzymes for the conversion of phosphoenolpyruvate to acetyl-CoA are not required to produce succinate at the theoretical yield in this example. Note that several other carbohydrates can be converted to

phosphoenolpyruvate and succinate by this simplified route (for example, compared to Figure 1) including xylose, arabinose, galactose, and glycerol.

[011] Figure 4 shows the reverse TCA cycle for fixation of C0 ₂ on carbohydrates as substrates. The enzymatic transformations are carried out by the enzymes as shown.

[012] Figure 5 shows the pathway for the reverse TCA cycle coupled with carbon monoxide dehydrogenase and hydrogenase for the conversion of syngas to acetyl-CoA.

[013] Figure 6 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M.

thermoacetica CODH (Moth_1202/1203) or Mtr (Moth l 197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng).

[014] Figure 7 shows CO oxidation assay results. Cells (M thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared. Assays were performed at 55°C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course. [015] Figure 8 A shows the nucleotide sequence (SEQ ID NO: 1) of carboxylic acid reductase from Nocardia iowensis (GNM_720), and Figure 8B shows the encoded amino acid sequence (SEQ ID NO:2).

[016] Figure 9A shows the nucleotide sequence (SEQ ID NO:3) of phosphpantetheine transferase, which was codon optimized, and Figure 9B shows the encoded amino acid sequence (SEQ ID NO:4).

[017] Figure 10A shows the nucleotide sequence (SEQ ID NO:5) of carboxylic acid reductase from Mycobacterium smegmatis mc(2)155 (designated 890), and Figure 10B shows the encoded amino acid sequence (SEQ ID NO:6).

[018] Figure 11A shows the nucleotide sequence (SEQ ID NO:7) of carboxylic acid reductase from Mycobacterium avium subspecies paratuberculosis K-10 (designated 891), and Figure 1 IB shows the encoded amino acid sequence (SEQ ID NO: 8).

[019] Figure 12A shows the nucleotide sequence (SEQ ID NO: 9) of carboxylic acid reductase from Mycobacterium marinum M (designated 892), and Figure 12B shows the encoded amino acid sequence (SEQ ID NO: 10).

[020] Figure 13A shows the nucleotide sequence (SEQ ID NO: 11) of carboxylic acid reductase designated 891GA, and Figure 13B shows the encoded amino acid sequence (SEQ ID NO: 12).

DETAILED DESCRIPTION OF THE INVENTION

[021] This invention is directed to the design and production of cells and organisms having increased production of succinate. In one embodiment, the invention utilizes gene additions and optionally gene deletions to increase production of succinate in a microbial host organism, in particular in bacteria or yeast. As disclosed herein, combinations of genetically engineering gene additions and/or strategically placed gene deletions or functional disruptions of genes can improve the succinate production capabilities of bacteria and other cells or organisms, including yeast. The microbial organisms and method disclosed herein are directed to increasing the production of succinate.

[022] Many organisms are capable of producing succinate. Examples of organisms innately capable of producing succinate from carbohydrates include Anaerobiospirillum succiniciproducens, Samuelov et al., Appl Environ Microbiol, 65: 2260-63 (1999), Lee et al., Appl Microbiol Biotechnol, 54: 23-27 (2000), Lee et al, Biotechnol Lett, 25: 111-14 (2003), Actinobacillus succinogenes, Guettler et al., Int J Syst Bacteriol, 49: 207-16 (1999), Urbance et al, Appl Microbiol Biotechnol, 65: 664-70 (2004), and the recently-sequenced bovine rumen bacterium, Mannheimia succiniciproducens, Lee et al., Bioprocess Biosyst Eng, 26: 63-7 (2003), Hong et al, Nat Biotechnol, 22: 1275-81 (2004), Lee et al, Appl Microbiol Biotechnol, 58: 663-8 (2002). In addition, some reports have purported to achieve the construction of Escherichia coli strains with improved succinate yields through various metabolic engineering strategies. These efforts have focused on increasing the channeling of carbon flux toward succinate and the availability of the cofactor NADH. For example, the overexpression of PEP carboxylase (ppc), Millard et al, Appl Environ Microbiol, 62: 1808- 10 (1996), and the expression of Rhizobium etli pyruvate carboxylase (pyc), Gokarn et al, Biotechnol Lett, 20: 795-798 (1998), have led to succinate yields in E. coli of 0.30 g/g and 0.17 g/g, respectively, by increasing the flux into the succinate branch of the TCA cycle. In addition, E. coli mutants deficient in lactate dehydrogenase (Idh) and pyruvate formate lyase (pfl) (i.e., strain NZNl 11), in conjunction with the overexpression of the E. coli, Stols et al, Appl Environ Microbiol, 63: 2695-701 (1997), Hong et al, Biotechnol Bioeng, 74: 89-95 (2001), or Ascaris suum, Stols et al, Appl Biochem Biotechnol, 63-65: 153-8 (1997), malic enzyme, have achieved improved succinate yields. An additional spontaneous chromosomal mutation in NZNl 11, later mapped to the ptsG gene of the phosphotransferase system, Chatterjee et al, Appl Environ Microbiol, 67: 148-54 (2001), led to strain AFPl 11 with an anaerobic succinate yield of 1 mol/mol glucose (0.66 g/g), Donnelly et al, Appl Biochem Biotechnol, 70-72: 187-98 (1998). Various properties of strains NZNl 11 and AFPl 11 in the presence and absence of the R. etli pyruvate carboxylase have been investigated under anaerobic and dual-phase conditions (i.e., aerobic growth followed by anaerobic production), Vemuri et al, Appl Environ Microbiol, 68: 1715-27, 18 (2002). Vemuri et al, J Ind

Microbiol Biotechnol, 28: 325-32 (2002), resulting in yields of about 0.96 g/g. Other efforts have resulted in succinate-producing strains of E. coli capable of achieving 0.91 mol/mol (0.60 g/g) aerobically, Lin et al, Metab Eng, (2005). In Press, and 1.6 mol/mol (1.0 g/g) anaerobically, Sanchez et al., Metab Eng, 7: 229-39 (2005).

[023] As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a succinate biosynthetic pathway.

[024] A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein.

[025] As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non- naturally occurring.

[026] As used herein, the terms "microbial," "microbial organism" or "microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. [027] As used herein, the term "succinate" is intended to mean the dicarboxylic acid HOOCCH ₂ CH ₂ COOH that is formed in the Krebs cycle and in various fermentation processes. The term "succinate" as it is used herein is synonymous with the term "succinic acid." Chemically, succinate corresponds to a salt or ester of succinic acid. Therefore, succinate and succinic acid refer to the same compound, which can be present in either of the two forms depending on the pH of the solution.

[028] As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system.

Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.

[029] As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen.

[030] "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid.

[031] It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

[032] As used herein, the term "gene disruption," or grammatical equivalents thereof, is intended to mean a genetic alteration that renders the encoded gene product inactive. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any of various mutation strategies that inactivate the encoded gene product. One particularly useful method of gene disruption is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the non- naturally occurring microorganisms of the invention.

[033] As used herein, the term "growth-coupled" when used in reference to the production of a biochemical product is intended to mean that the biosynthesis of the referenced biochemical product is produced during the growth phase of a microorganism. In a particular embodiment, the growth-coupled production can be obligatory, meaning that the biosynthesis of the referenced biochemical is an obligatory product produced during the growth phase of a microorganism.

[034] The non-naturally occurring microbal organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.

[035] In the case of gene disruptions, a particularly useful stable genetic alteration is a gene deletion. The use of a gene deletion to introduce a stable genetic alteration is particularly useful to reduce the likelihood of a reversion to a phenotype prior to the genetic alteration. For example, stable growth-coupled production of a biochemical can be achieved, for example, by deletion of a gene encoding an enzyme catalyzing one or more reactions within a set of metabolic modifications. The stability of growth-coupled production of a biochemical can be further enhanced through multiple deletions, significantly reducing the likelihood of multiple compensatory reversions occurring for each disrupted activity.

[036] Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.

However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements. [037] An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

[038] Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5 '-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

[039] In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

[040] A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

[041] Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having succinate biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly for a gene disruption, evolutionally related genes can also be disrupted or deleted in a host microbial organism to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption.

[042] Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

[043] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept- 16- 1998) and the following parameters: Match: 1;

mismatch: -2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

[044] The invention is directed to microbial organisms having increased production of succinate. Since many organisms are naturally capable of producing succinate (see above), it is understood that the microbial organisms and methods of using such organisms as disclosed herein, when referring to succinate production, refers to an increase in production relative to succinate production in the absence of the genetic modificiations described herein as providing an increase in succinate production.

[045] In one embodiment, the invention provides a non-naturally occurring microbial organism, where the microbial organism comprises a genetic modification, for example, one or more genetic modifications, where the genetic modification confers to the microbial organism increased production of succinate relative to the microbial organism in the absence of the genetic modification. Such a non-naturally occurring microbial organism can comprise (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein the at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein the at least one exogenous nucleic acid is selected from a pyruvate :ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H ₂ hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H ₂ hydrogenase, and combinations thereof. It is understood that a genetic modification can be selected from those provide in (i), (ii) or (iii), which result in increased succinate production, or the genetic modification can be a separate genetic modification or modifications which, on its own, provides increased production of succinate. In such a case, the further modifications with (i), (ii) and (iii) provide a further increase in succinate production.

[046] In a particular embodiment, the a genetic modification can comprise expression of at least one exogenous nucleic acid encoding an enzyme selected from an enzyme shown in Figures 1, 2 or 3. In another embodiment, a microbial organism comprising (i) can further comprise an exogenous nucleic acid encoding an enzyme selected from a pyruvate :ferredoxin oxidoreductase, an aconitase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In addition, a microbial organism comprising (ii) can further comprise an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl- CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, NAD(P):ferredoxin oxidoreductase, ferredoxin, and combinations thereof. Further, a microbial organism comprising (iii) can further comprise an exogenous nucleic acid encoding an enzyme selected from NAD(P): ferredoxin oxidoreductase and ferredoxin.

[047] Exemplary enzymes shown in Figure 1 include pyruvate kinase;

phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase; malic enzyme; pyruvate carboxylase; pyruvate carboxylase; pyruvate dehydrogenase or pyruvate formate lyase and formate dehydrogenase; citrate synthase; aconitase; isocitrate dehydrogenase; alpha-ketoglutarate dehydrogenase; succinyl-CoA ligase, succinyl-CoA transferase or succinyl-coA hydrolase; malate dehydrogenase; fumarase; fumarate reductase; isocitrate lyase; and malate synthase. Exemplary enzymes shown in Figure 2 include pyruvate kinase; phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase; malic enzyme; pyruvate carboxylase; pyruvate: ferredoxin oxidoreductase or pyruvate formate lyase and formate dehydrogenase; acetyl-CoA synthase or acetate kinase and phosphotransacetylase; citrate lyase; aconitase; isocitrate dehydrogenase; alpha-ketoglutarate ferredoxin

oxidoreductase; succinyl-CoA transferase or succinyl-CoA synthetase; ATP-citrate lyase; malate dehydrogenase; fumarase; fumarate reductase; isocitrate lyase; and malate synthase. Exemplary enzymes shown in Figure 3 include pyruvate kinase; phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase; malic enzyme; pyruvate carboxylase; malate dehydrogenase; fumarase; and fumarate reductase. In a particular embodiment, the enzyme is selected from phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase; malic enzyme; pyruvate carboxylase; isocitrate lyase; and malate synthase.

[048] The invention additionally provides a microbial organism comprising two, three, four or five exogenous nucleic acids each encoding enzymes of (i), (ii) or (iii). For example, a microbial organism comprising (i) can comprise three exogenous nucleic acids encoding ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, or a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate: ferredoxin oxidoreductase; a microbial organism comprising (ii) can comprise five exogenous nucleic acids encoding pyruvate: ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H ₂ hydrogenase; or a microbial organism comprising (iii) can comprise two exogenous nucleic acids encoding CO dehydrogenase and H ₂ hydrogenase. In a further embodiment, a microbial organism comprising (ii) or (iii) can further comprise an exogenous nucleic acid encoding an enzyme selected from NAD(P):ferredoxin oxidoreductase and ferredoxin.

[049] The invention additionally provides a non-naturally occurring microbial organism having increased production of succinate, as described above and herein, where the microbial organism further comprises one or more gene disruptions. The one or more gene disruptions occur in genes encoding proteins or enzymes where the one or more gene disruptions confer increased production of succinate in the organism relative to the absence of said gene disruptions. Such a gene disruption is therefore considered to be a genetic modification conferring increased production of succinate. As discussed above, such a genetic

modification can be combined with a reductive TCA pathwy and/or CO dehydrogenase and/or H ₂ hydrogenase to further increase production of succinate. In such a microbial organism with a gene disruption, the production of succinate can be growth-coupled or not growth coupled.

[050] Such a microbial organism can comprise one or more gene disruptions of a gene encoding a protein or enzyme selected from pyruvate kinase, alcohol dehydrogenase; lactate dehydrogenase; acetate kinase, pyruvate oxidase, pyruvate formate lyase and

phosphotransferase system of glucose transport. Examples of such genes include adhE, IdhA, ackA, poxB, pflB, tdcE, iclR, pstG, ptsHl and err. In another ebmodiment, a microbial organism of the invention can further comprise a genetic modification comprising expression of at least one exogenous nucleic acid encoding an enzyme or protein selected from glucokinase and galactose permease. As disclosed herein, a gene disruption can be deletion of the one or more disrupted genes.

[051] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a succinate pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of

phosphoenolpyruvate to oxaloacetate; phosphoenolpyruvate to malate; pyruvate to

oxaloacetate; oxaloacetate to malate; malate to fumarate; and fumarate to succinate (see Figures 1-3). Additionally, a microbial organism can comprise at least one exogenous nucleic acid that converts acetyl-CoA and oxaloacetate to citrate; citrate to D-isocitrate; D- isocitrate to alpha-ketoglutarate; alpha-ketoglutarate to succinyl-CoA, and succinyl-CoA to succinate (see Figure 1). Optionally such a microbial organism can further comprise at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from D-isocitrate to glyoxylate or succinate; and glyoxylate to malate (Figure 1). Further, a microbial organism can comprise at least one exogenous nucleic acid that converts succinate to succinyl-CoA; succinyl-CoA to alpha-ketoglutarate; alpha- ketoglutarate to D-isocitrate; D-isocitrate to citrate; citrate to oxaloacetate or to acetate;

acetate to acetyl-CoA; and acetyl-CoA to pyruvate (see Figure 2). One skilled in the art will understand that these are merely exemplary and that any of the substrate -product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a succinate pathway, such as that shown in Figures 1-3.

[052] While generally described herein as a microbial organism that contains a succinate pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a succinate pathway enzyme expressed in a sufficient amount to produce an intermediate of a succinate pathway. For example, as disclosed herein, succinate pathways are exemplified in Figures 1- 3. Therefore, in addition to a microbial organism containing a succinate pathway that produces succinate, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a succinate pathway enzyme, where the microbial organism produces a succinate pathway intermediate, for example, oxaloacetate; malate, fumarate, citrate, D-isocitrate, alpha-ketoglutarate, succinyl- CoA, glyoxylate, acetate, or acetyl-CoA (see Figures 1-3).

[053] It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 1-3, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a succinate pathway intermediate can be utilized to produce the intermediate as a desired product. [054] This invention is also directed, in part to engineered biosynthetic pathways to improve carbon flux through a central metabolism intermediate en route to succinate. The present invention provides non-naturally occurring microbial organisms having one or more exogenous genes encoding enzymes that can catalyze various enzymatic transformations en route to succinate. In some embodiments, these enzymatic transformations are part of the reductive tricarboxylic acid (RTCA) cycle and are used to improve product yields, including but not limited to, from carbohydrate-based carbon feedstock.

[055] In numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents and/or carbon to byproducts. In accordance with some embodiments, the present invention increases the yields of succinate by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C0 ₂ , and/or H ₂ . In addition to syngas, other sources of such gases include, but are not limted to, the atmosphere, either as found in nature or generated.

[056] The C0 ₂ -fixing reductive tricarboxylic acid (RTCA) cycle is an endergenic anabolic pathway of C0 ₂ assimilation which uses reducing equivalents and ATP (Figure 4). One turn of the RTCA cycle assimilates two moles of C0 ₂ into one mole of acetyl-CoA, or four moles of C0 ₂ into one mole of oxaloacetate. This additional availability of acetyl-CoA improves the maximum theoretical yield of product molecules derived from carbohydrate- based carbon feedstock. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose and glycerol.

[057] In some embodiments, the reductive TCA cycle, coupled with carbon monoxide dehydrogenase and/or hydrogenase enzymes, can be employed to allow syngas, C0 ₂ , CO, H ₂ , and/or other gaseous carbon source utilization by microorganisms. Synthesis gas (syngas), in particular is a mixture of primarily H ₂ and CO, sometimes including some amounts of C0 ₂ , that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter. Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids full combustion, of organic materials at high temperatures (500-1500°C) to provide syngas as a 0.5: 1-3: 1 H ₂ /CO mixture. In addition to coal, biomass of many types has been used for syngas production and represents an inexpensive and flexible feedstock for the biological production of renewable chemicals and fuels. Carbon dioxide can be provided from the atmosphere or in condensed from, for example, from a tank cylinder, or via sublimation of solid C0 ₂ . Similarly, CO and hydrogen gas can be provided in reagent form and/or mixed in any desired ratio. Other gaseous carbon forms can include, for example, methanol or similar volatile organic solvents.

[058] The components of synthesis gas and/or other carbon sources can provide sufficient C0 ₂ , reducing equivalents, and ATP for the reductive TCA cycle to operate. One turn of the RTCA cycle assimilates two moles of C0 ₂ into one mole of acetyl-CoA and requires 2 ATP and 4 reducing equivalents. CO and/or H ₂ can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase enzymes, respectively. Reducing equivalents can come in the form of NADH, NADPH, FADH, reduced quinones, reduced ferredoxins, reduced flavodoxins and thioredoxins. The reducing equivalents, particularly NADH, NADPH, and reduced ferredoxin, can serve as cofactors for the RTCA cycle enzymes, for example, malate dehydrogenase, fumarate reductase, alpha- ketoglutarate: ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate: ferredoxin oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase),

pyruvate: ferredoxin oxidoreductase and isocitrate dehydrogenase. The electrons from these reducing equivalents can alternatively pass through an ion-gradient producing electron transport chain where they are passed to an acceptor such as oxygen, nitrate, oxidized metal ions, protons, or an electrode. The ion-gradient can then be used for ATP generation via an ATP synthase or similar enzyme.

[059] The reductive TCA cycle was first reported in the green sulfur photosynthetic bacterium Chlorobium limicola (Evans et al, Proc. Natl. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways have been characterized in some prokaryotes (proteobacteria, green sulfur bacteria and thermophillic Knallgas bacteria) and sulfur-dependent archaea (Hugler et al, J. Bacteriol. 187:3020-3027 (2005; Hugler et al, Environ. Microbiol. 9:81-92 (2007). In some cases, reductive and oxidative (Krebs) TCA cycles are present in the same organism (Hugler et al, supra (2007); Siebers et al, J. Bacteriol. 186:2179-2194 (2004)). Some methanogens and obligate anaerobes possess incomplete oxidative or reductive TCA cycles that may function to synthesize biosynthetic intermediates (Ekiel et al., J. Bacteriol. 162:905-908 (1985); Wood et al, F EMS Microbiol. Rev. 28:335-352 (2004)). [060] The key carbon-fixing enzymes of the reductive TCA cycle are alpha- ketoglutarate:ferredoxin oxidoreductase, pyruvate :ferredoxin oxidoreductase and isocitrate dehydrogenase. Additional carbon may be fixed during the conversion of

phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxylase or

phosphoenolpyruvate carboxykinase or by conversion of pyruvate to malate by malic enzyme.

[061] Many of the enzymes in the TCA cycle are reversible and can catalyze reactions in the reductive and oxidative directions. However, some TCA cycle reactions are irreversible in vivo and thus different enzymes are used to catalyze these reactions in the directions required for the reverse TCA cycle. These reactions are: (1) conversion of citrate to oxaloacetate and acetyl-CoA, (2) conversion of fumarate to succinate, and (3) conversion of succinyl-CoA to alpha-ketoglutarate. In the TCA cycle, citrate is formed from the condensation of oxaloacetate and acetyl-CoA. The reverse reaction, cleavage of citrate to oxaloacetate and acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase, or citryl- CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyase can be coupled to acetyl- CoA synthetase, an acetyl-CoA transferase, or phosphotransacetylase and acetate kinase to form acetyl-CoA and oxaloacetate from citrate. The conversion of succinate to fumarate is catalyzed by succinate dehydrogenase while the reverse reaction is catalyzed by fumarate reductase. In the TCA cycle succinyl-CoA is formed from the NAD(P) ⁺ dependent decarboxylation of alpha-ketoglutarate by the alpha-ketoglutarate dehydrogenase complex. The reverse reaction is catalyzed by alpha-ketoglutarate :ferredoxin oxidoreductase.

[062] An organism capable of utilizing the reverse tricarboxylic acid cycle to enable production of acetyl-CoA-derived products on 1) CO, 2) C0 ₂ and H ₂ , 3) CO and C0 ₂ , 4) synthesis gas comprising CO and H ₂ , and 5) synthesis gas or other gaseous carbon sources comprising CO, C0 ₂ , and H ₂ can include any of the following enzyme activities: ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate :ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, acetate kinase, phosphotransacetylase, acetyl-CoA synthetase, acetyl-CoA transferase,

pyruvate :ferredoxin oxidoreductase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, hydrogenase, and ferredoxin (see Figure 5). Enzyme and the corresponding genes required for these activities are described herein below. [063] Carbon from syngas or other gaseous carbon sources can be fixed via the reverse TCA cycle and components thereof. Specifically, the combination of certain carbon gas- utilization pathway components with the pathways for formation of succinate from acetyl- CoA results in high yields of these products by providing an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogenously or produced endogenously from CO, into acetyl-CoA.

[064] In some embodiments, a succinate pathway in a non-naturally occurring microbial organism of the invention can utilize any combination of (1) CO, (2) C0 ₂ , (3) H ₂ , or mixtures thereof to enhance the yields of biosynthetic steps involving reduction, including addition to driving the reductive TCA cycle.

[065] In some embodiments a non-naturally occurring microbial organism having a succinate pathway includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; and at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C0 ₂ , (3) H ₂ , (4) C0 ₂ and H ₂ , (5) CO and C0 ₂ , (6) CO and H ₂ , or (7) CO, C0 ₂ , and H ₂ .

[066] In some embodiments a method includes culturing a non-naturally occurring microbial organism having a succinate pathway also comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha- ketoglutarate: ferredoxin oxidoreductase. Additionally, such an organism can also include at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a

hydrogenase, a NAD (P)H: ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C0 ₂ , (3) H ₂ , (4) C0 ₂ and H ₂ , (5) CO and C0 ₂ , (6) CO and H ₂ , or (7) CO, C0 ₂ , and H ₂ to produce a product.

[067] In some embodiments a non-naturally occurring microbial organism having a succinate pathway further includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, a pyruvate :ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase, and an alpha- ketoglutarate : ferredoxin oxidoreductase .

[068] In some embodiments a non-naturally occurring microbial organism having a succinate pathway includes at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of carbon monoxide and/or hydrogen, thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock. The at least one exogenous nucleic acid is selected from a carbon monoxide dehydrogenase, a hydrogenase, an NAD(P)H: ferredoxin oxidoreductase, and a ferredoxin. In some embodiments, the present invention provides a method for enhancing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock, such as sugars or gaseous carbon sources, the method includes culturing this non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce succinate.

[069] In some embodiments, the non-naturally occurring microbial organism having a succinate pathway includes two exogenous nucleic acids, each encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism having a succinate pathway includes three exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism includes three exogenous nucleic acids encoding an ATP-citrate lyase, or optionally a citryl-CoA synthetase and/or a citryl-CoA lyase, a fumarate reductase, and an alpha-ketoglutarate: ferredoxin oxidoreductase. In some embodiments, the non-naturally occurring microbial organism includes three exogenous nucleic acids encoding a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate :ferredoxin oxidoreductase.

[070] In some embodiments, the non-naturally occurring microbial organisms having a succinate pathway further include an exogenous nucleic acid encoding an enzyme selected from a pyruvate: ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an

NAD(P)H:ferredoxin oxidoreductase, and combinations thereof.

[071] In some embodiments, the non-naturally occurring microbial organism having a succinate pathway further includes an exogenous nucleic acid encoding an enzyme selected from carbon monoxide dehydrogenase, acetyl-CoA synthase, ferredoxin,

NAD(P)H:ferredoxin oxidoreductase and combinations thereof. In some embodiments, the non-naturally occurring microbial organism having a succinate pathway utilizes a carbon feedstock selected from (1) CO, (2) C0 ₂ , (3) C0 ₂ and H ₂ , (4) CO and H ₂ , or (5) CO, C0 ₂ , and H ₂ . In some embodiments, the non-naturally occurring microbial organism having a succinate pathway utilizes hydrogen for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having a succinate pathway utilizes CO for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having a succinate pathway utilizes combinations of CO and hydrogen for reducing equivalents.

[072] In some embodiments, the non-naturally occurring microbial organism having a succinate pathway further includes one or more nucleic acids encoding an enzyme selected from a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a pyruvate carboxylase, and a malic enzyme. In some embodiments, the non-naturally occurring microbial organism having a succinate pathway further includes one or more nucleic acids encoding an enzyme selected from a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA synthetase, and a succinyl-CoA transferase. In some

embodiments, the non-naturally occurring microbial organism having succinate pathway further includes at least one exogenous nucleic acid encoding a citrate lyase, an ATP-citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, and a ferredoxin.

[073] It is understood by those skilled in the art that the above-described pathways for increasing product yield can be combined with any of the pathways disclosed herein, including those pathways depicted in the figures. One skilled in the art will understand that, depending on the pathway to a desired product and the precursors and intermediates of that pathway, a particular pathway for improving product yield, as discussed herein above and in the examples, or combination of such pathways, can be used in combination with a pathway to a desired product to increase the yield of that product or a pathway intermediate.

[074] The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

[075] As disclosed herein, the product succinate or other carboxylic acids, as well as other intermediates, are carboxylic acids, which can occur in various ionized forms, including fully protonated, partially protonated, and fully deprotonated forms. Accordingly, the suffix "-ate," or the acid form, can be used interchangeably to describe both the free acid form as well as any deprotonated form, in particular since the ionized form is known to depend on the pH in which the compound is found. It is understood that carboxylate products or

intermediates includes ester forms of carboxylate products or pathway intermediates, such as O-carboxylate and S-carboxylate esters. O- and S-carboxylates can include lower alkyl, that is CI to C6, branched or straight chain carboxylates. Some such O- or S-carboxylates include, without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert- butyl, pentyl, hexyl O- or S-carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, and hexenyl O- or S-carboxylates. O- carboxylates can be the product of a biosynthetic pathway. Exemplary O-carboxylates accessed via biosynthetic pathways can include, without limitation, methyl succinate, ethyl succinate, and n-propyl succinate. Other bio synthetically accessible O-carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such hexyl, heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations. O-Carboxylate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an O- or S- carboxylate. S-Carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters.

[076] The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more succinate biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular succinate biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve succinate biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as succinate.

[077] Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobio spirillum

succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens,

Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.

Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae,

Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae and Candida. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.

[078] Depending on the succinate biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed succinate pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more succinate biosynthetic pathways. For example, succinate biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a succinate pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of succinate can be included, if desired. For example, exogenous expression can include the enzymes of the pathways as shown in Figures 1-3.

[079] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the succinate pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, or more, as desired, including up to all or a subset of all nucleic acids encoding the enzymes or proteins constituting a succinate biosynthetic pathway disclosed herein, for example, as shown in Figures 1-3. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize succinate biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the succinate pathway precursors such as or disruption of a gene, both of which increase the production of succinate.

[080] Generally, a host microbial organism is selected such that it produces the precursor of a succinate pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, malate is produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a succinate pathway.

[081] In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize succinate. In this specific embodiment it can be useful to increase the synthesis or accumulation of a succinate pathway product to, for example, drive succinate pathway reactions toward succinate production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described succinate pathway enzymes or proteins. Overexpression of the enzyme or enzymes and/or protein or proteins of the succinate pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing succinate, through overexpression of one, two, three, four, five, six, seven, eight or more, that is, up to all nucleic acids encoding succinate biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the succinate biosynthetic pathway.

[082] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

[083] It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non- naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a succinate biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer succinate biosynthetic capability. For example, a non-naturally occurring microbial organism having a succinatebiosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of malic enzyme and fumarate reductase; phosphoenolpyruvate carboxylase and glyoxylate, and so forth (see Figures 1-3). Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, for example, phosphoenolpyruvate carboxykinase, malate dehydrogenase and fumarate reductase; pyruvate carboxylase, fumarate reductase and malate dehydrogenase; isocitrate lyase, malate synthase and fumarase and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of four, fumarase, isocitrate lyase, malate dehydrogenase and fumarate reductase;

phosphoenolpyruvate carboxylase, pyruvate carboxylase, isocitrate lyase and fumarase, or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

[084] In addition to the biosynthesis of succinate as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce succinate other than use of the succinate producers is through addition of another microbial organism capable of converting a succinate pathway intermediate to succinate. One such procedure includes, for example, the fermentation of a microbial organism that produces a succinate pathway intermediate. The succinate pathway intermediate can then be used as a substrate for a second microbial organism that converts the succinate pathway intermediate to succinate. The succinate pathway intermediate can be added directly to another culture of the second organism or the original culture of the succinate pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.

[085] In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, succinate. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of succinate can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, succinate also can be biosynthetically produced from microbial organisms through co-culture or co -fermentation using two organisms in the same vessel, where the first microbial organism produces a succinate intermediate and the second microbial organism converts the intermediate to succinate.

[086] Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce succinate.

[087] Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase production of succinate. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods of the invention can be applied to any suitable host

microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase succinate biosynthesis. In a particular embodiment, the increased production couples biosynthesis of succinate to growth of the organism, and can obligatorily couple production of succinate to growth of the organism if desired and as disclosed herein.

[088] Sources of encoding nucleic acids for a succinate pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Saccharomyces cerevisiae,

Acinetobacter baumannii, Pseudomonas aeruginosa, Chlorobium limicola, Chlorobium tepidum, Balnearium lithotrophicum, Sulfurihydrogenibium subterraneum, Sulfurimonas denitrificans, Yarrowia lipolytica, Schizosaccharomyces pombe, Sordaria macrospora, Aspergillus nidulans, Hydrogenobacter thermophilus, Aquifex aeolicus, Corynebacterium glutamicum, Campylobacter jejuni, Thermus thermophilus, Rattus norvegicus,

Pelotomaculum thermopropionicum, Moorella thermoacetica, Sulfolobus, Aeropyrum pernix, Helicobacter pylori, Thauera aromatica, Rhodospirillum rubrum, Geobacter sulfurreducens, Sulfurimonas denitrificans, Thiobacillus denitrificans, Thermocrinis albus, Ralstonia eutropha, Desulfovibrio fructosovorans, Sulfurimonas denitrificans, Salmonella typhimurium, Desulfovibrio africanus, Desulfovibrio vulgaris , Desulfovibrio desulfuricans , Rhodobacter capsulatus, Clostridium kluyveri, Rhodopseudomonas palustris, Bacillus subtilis, Zea mays, Clostridium carboxidivorans , Clostridium pasteurianum, Sulfolobus acidocalarius,

Carboxydothermus hydrogenoformans, Rhodobacter capsulatus, Allochromatium vinosum , Azotobacter vinelandii , Rhodopseudomonas palustris, Thauera aromatica,

Carboxydothermus hydrogenoformans, Pseudomonas aeruginosa , Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas putida, Acetobacter aceti, Homo sapiens, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Azoarcus, Aromatoleum aromaticum, Geobacter metallireducens, Citrobacter youngae, Salmonella enterica, Yersinia intermedia, Leuconostoc mesenteroides, Klebsiella pneumoniae, Methanosarcina

thermophila, Thermotoga maritima, butyrate-producing bacterium L2-50, Bacillus megaterium, Methanothermobacter thermautotrophicus, Salmonella enterica, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Pseudomonas putida,

Carboxydothermus hydrogenoformans, Clostridium carboxidivorans, Geobacter

metallireducens, Chlorobium phaeobacteroides, Clostridium cellulolyticum , Desulfovibrio desulfuricans, Pelobacter carbinolicus , Campylobacter curvus, Carboxydothermus hydrogenoformans, Rhodospirillum rubrum, Geobacter sulfurreducens, Synechocystis species, Nostoc species , Thiocapsa roseopersicina, Methylobacterium extorquens,

Corynebacterium glutamicum, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Haemophilus influenza, Mycobacterium smegmatis, Ascaris suum, Nocardia iowensis, Mycobacterium bovis, Nocardia farcinica , Streptomyces griseus , Mycobacterium smegmatis , Mycobacterium avium, Mycobacterium marinum, Tsukamurella paurometabola, Cyanobium, Dictyostelium discoideum, Candida albicans, Penicillium chrysogenum

[089] as well as other exemplary species disclosed herein (see Examples) or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite succinate biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of succinate described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms. [090] In some instances, such as when an alternative succinate biosynthetic pathway exists in an unrelated species, succinate biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a species of interest that will synthesize succinate.

[091] Methods for constructing and testing the expression levels of a non-naturally occurring succinate-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[092] Exogenous nucleic acid sequences involved in a pathway for production of succinate can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al, J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

[093] An expression vector or vectors can be constructed to include one or more succinate biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or

immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[094] The invention additionally provides methods for producing succinate utilizing the succinate producing microbial organisms of the invention, as described herein. The methods involve culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce succinate. The microbial organism can be provided, for example, in a substantially anaerobic culture medium.

[095] Suitable purification and/or assays to test for the production of succinate can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al, Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art.

[096] The succinate product can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

[097] Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the succinate producers can be cultured for the biosynthetic production of succinate.

[098] For the production of succinate, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art.

Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high succinate yields.

[099] If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

[0100] The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of succinate.

[0101] In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in succinate or any succinate pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product succinate or succinate pathway intermediate, or for side products generated in reactions diverging away from a succinate pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

[0102] In some embodiments, the uptake sources can be selected to alter the carbon- 12, carbon- 13, and carbon- 14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen- 16, oxygen- 17, and oxygen- 18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can be selected to alter the nitrogen- 14 and nitrogen- 15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur- 33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31 , phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios.

[0103] In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially derived from a chemical synthetic reaction. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to achieve a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a desired origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a biological organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel- derived carbon source, which can be relatively depleted of carbon- 14, or an environmental or atmospheric carbon source, such as C0 ₂ , which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

[0104] The unstable carbon isotope carbon- 14 or radiocarbon makes up for roughly 1 in 10 ¹² carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen ( ¹⁴ N). Fossil fuels contain no carbon-14, as it decayed long ago. Burning of fossil fuels lowers the atmospheric carbon- 14 fraction, the so-called "Suess effect".

[0105] Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass

spectrometry using techniques known in the art such as accelerated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like.

[0106] In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011).

Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.

[0107] The biobased content of a compound is estimated by the ratio of carbon- 14 ( ¹⁴ C) to carbon-12 ( ¹² C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S-B)/(M-B), where B, S and M represent the ¹⁴ C/ ¹² C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹⁴ C/ ¹² C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard reference materials (SRM) 4990b) normalized to per mil (Olsson, The use of Oxalic acid as a Standard, in, Radiocarbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc, John Wiley & Sons, New York (1970)). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalized to 5 ¹³ C _VPDB =-19 per mil. This is equivalent to an absolute (AD 1950) ¹⁴ C/ ¹² C ratio of 1.176 ± 0.010 x 10 ^"12 (Karlen et al, Arkiv Geofysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one istope with respect to another, for example, the preferential uptake in biological systems of C over C over C , and these corrections are reflected as a Fm corrected for δ ¹³ .

[0108] An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially available. The Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). The isotopic ratio of HOx II is -17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid II standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently available oxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm = 0% represents the entire lack of carbon- 14 atoms in a material, thus indicating a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post-1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a "modern" source includes biobased sources.

[0109] As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but diminishing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of carbon-14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a "pre-bomb" standard, and because nearly all new biobased products are produced in a post- bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true biobased content of the sample. A biobased content that is greater than 103% suggests that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

[0110] ASTM D6866 quantifies the biobased content relative to the material's total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50%> starch-based material and 50%> water would be considered to have a Biobased Content = 100% (50%> organic content that is 100%) biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a Biobased Content = 66.7%> (75%o organic content but only 50%> of the product is biobased). In another example, a product that is 50%> organic carbon and is a petroleum-based product would be considered to have a Biobased Content = 0% (50% organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one skilled in the art can readily determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content.

[0111] Applications of carbon- 14 dating techniques to quantify bio-based content of materials are known in the art (Currie et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon- 14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable 1,3-propanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) (Currie et al., supra, 2000). In contrast, polybutylene terephthalate polymer derived from both renewable 1 ,4-butanediol and renewable terephthalic acid resulted in bio-based content exceeding 90% (Colonna et al., supra, 2011).

[0112] Accordingly, in some embodiments, the present invention provides succinate or a succinate pathway intermediate that has a carbon- 12, carbon- 13, and carbon- 14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the succinate or a succinate pathway intermediate can have an Fm value of at least 10%>, at least 15%>, at least 20%>, at least 25%>, at least 30%>, at least 35%>, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%). In some such embodiments, the uptake source is C0 ₂ . In some embodiments, the present invention provides succinate or a succinate pathway intermediate that has a carbon- 12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the succinate or a succinate intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%>, less than 25%>, less than 20%>, less than 15%>, less than 10%>, less than 5%>, less than 2% or less than 1%. In some embodiments, the present invention provides succinate or a succinate pathway intermediate that has a carbon- 12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake sources is one way by which the carbon- 12, carbon- 13, and carbon- 14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

[0113] Further, the present invention relates to the biologically produced succinate or succinate pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the succinate or a succinate pathway intermediate has a carbon- 12, carbon- 13, and carbon- 14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment. For example, in some aspects the invention provides bioderived succinate or a bioderived succinate pathway intermediate having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as disclosed herein, that a product can have a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from bioderived succinate or a bioderived succinate pathway intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of succinate, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides products made or derived from succinate, including but not limited to butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polymers such as polybutylene succinate polymers, which can be used as a biodegreadable plastic to replace conventional plastics in applications such as flexible packaging, agricultural films and compostable bags; a combination of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers for applications such as automotive interiors, nonwovens, construction materials and consumer goods, and the like, having a carbon- 12 versus carbon- 13 versus carbon- 14 isotope ratio of about the same value as the C0 ₂ that occurs in the environment, wherein the butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco- valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like, are generated directly from or in combination with bioderived succinate or a bioderived succinate pathway intermediate as disclosed herein.

[0114] Succinate is a chemical used in commercial and industrial applications. Non- limiting examples of such applications include production of butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco- valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like. Moreover, succinate is also used as a raw material in the production of a wide range of products including butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like. Accordingly, in some embodiments, the invention provides biobased butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating;

polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like, comprising one or more bioderived succinate or bioderived succinate pathway intermediates produced by a non- naturally occurring microorganism of the invention or produced using a method disclosed herein.

[0115] As used herein, the term "bioderived" means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can utilize feedstock or biomass, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source. Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term "biobased" means a product as described above that is composed, in whole or in part, of a bioderived compound of the invention. A biobased or bioderived product is in contrast to a petroleum derived product, wherein such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

[0116] In some embodiments, the invention provides butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco- valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like, comprising bioderived succinate or a bioderived succinate pathway intermediate, wherein the bioderived succinate or bioderived succinate pathway intermediate includes all or part of the succinate or succinate pathway intermediate used in the production of butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco- valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like. Thus, in some aspects, the invention provides a biobased butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors,

nonwovens, construction materials and consumer goods, and the like, comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%), at least 98%> or 100% bioderived succinate or bioderived succinate pathway intermediate as disclosed herein. Additionally, in some aspects, the invention provides a biobased butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like, wherein the succinate or succinate pathway intermediate used in its production is a combination of bioderived and petroleum derived succinate or succinate pathway intermediate. For example, a biobased butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like, can be produced using 50%> bioderived succiante and 50%> petroleum derived succinate or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed herein. It is understood that methods for producing butanediol, tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics, fuel additives, fabrics, carpets, pigments, detergents, metal plating; polybutylene succinate polymers, biodegreadable plastics, flexible packaging, agricultural films, compostable bags, combinations of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plasitics such as polylactic acid, polyhydroxyalkanoate, and poly-3 -hydroxy butyrateco-valerate; and a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials and consumer goods, and the like, using the bioderived succinate or a bioderived succinate pathway intermediate of the invention are well known in the art.

[0117] In addition to renewable feedstocks such as those exemplified above, the succinate-producing microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the succinate producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

[0118] Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H ₂ and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H ₂ and CO, syngas can also include C0 ₂ and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C0 ₂ .

[0119] The Wood-Ljungdahl pathway catalyzes the conversion of CO and H ₂ to acetyl- CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing C0 ₂ and C0 ₂ /H ₂ mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. Independent conversion of C0 ₂ to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of C0 ₂ and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

2 C0 ₂ + 4 H ₂ + n ADP + n Pi→ CH ₃ COOH + 2 H ₂ 0 + n ATP Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize C0 ₂ and H ₂ mixtures as well for the production of acetyl-CoA and other desired products.

[0120] The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase,

methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolatexorrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide

dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a succinate pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

[0121] Additionally, as disclosed herein the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, C0 ₂ and/or H ₂ to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, a citryl-CoA synthetase, a citryl- CoA lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate: ferredoxin

oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H: ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H ₂ by carbon monoxide dehydrogenase and hydrogenase are utilized to fix C0 ₂ via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl- CoA synthetase. Acetyl-CoA can be converted to the succinate precursors, glyceraldehyde- 3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate :ferredoxin oxidoreductase and the enzymes of gluconeo genesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a succinate pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete reductive TCA pathway will confer syngas utilization ability.

[0122] Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, succinate and any of the intermediate metabolites in the succinate pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the succinate biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes succinate when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the succinate pathway when grown on a carbohydrate or other carbon source. The succinate producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, oxaloacetate, malate, fumarate, and the like (see Figures 1-3).

[0123] The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a succinate pathway enzyme or protein in sufficient amounts to produce succinate. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce succinate. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of succinate resulting in intracellular concentrations between about 0.1- 200 mM or more. Generally, the intracellular concentration of succinate is between about 3- 150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above each of these exemplary ranges also can be achieved from the non- naturally occurring microbial organisms of the invention.

[0124] In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S.

publication 2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the succinate producers can synthesize succinate at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, succinate producing microbial organisms can produce succinate intracellularly and/or secrete the product into the culture medium.

[0125] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of succinate can include the addition of an

osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2- methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about lOOmM or no more than about 500mM.

[0126] The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

[0127] As described herein, one exemplary growth condition for achieving biosynthesis of succinate includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N ₂ /CO ₂ mixture or other suitable non-oxygen gas or gases.

[0128] The culture conditions described herein can be scaled up and grown continuously for manufacturing of succinate. Exemplary growth procedures include, for example, fed- batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of succinate. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of succinate will include culturing a non-naturally occurring succinate producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

[0129] Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of succinate can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[0130] In addition to the above fermentation procedures using the succinate producers of the invention for continuous production of substantial quantities of succinate, the succinate producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical or enzymatic conversion to convert the product to other compounds, if desired.

[0131] To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US

2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of succinate.

[0132] One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al, Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth- coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.

[0133] Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007.

[0134] Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003. SimPheny® is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

[0135] These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways.

Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted.

[0136] Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art.

[0137] The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes. [0138] Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.

[0139] To identify additional productive solutions to the above described bilevel

OptKnock problem which lead to further sets of reactions to disrupt or metabolic

modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

[0140] The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

[0141] As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al, Biotechnol. Bioeng. 84:647-657 (2003)).

[0142] An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US

2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US

2004/0009466, and in U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth- coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above.

[0143] Employing the methods exemplified above, the methods of the invention allow the construction of cells and organisms that increase production of a desired product, for example, by engineering a cell or organism to incorporate a genetic modification of a gene disruption of one or more genes that result in an increase in production of succinate. In one embodiment, the production of a desired product such as succinate is not coupled to growth. In another embodiment, the production of a desired product such as succinate is coupled to growth of the cell or organism engineered to harbor the identified genetic alterations. As disclosed herein, disruption of certain genes can lead to an increase in production of succinate (see Examples). Microbial organism strains constructed with the identified metabolic alterations produce elevated levels, relative to the absence of the metabolic alterations, of succinate. If desired, the production of succinate can be increased during the exponential growth phase. Strains having growth coupled production can be used for the commercial production of succinate in continuous fermentation process without being subjected to the negative selective pressures described previously. Although exemplified herein as metabolic alterations, in particular one or more gene disruptions, that confer growth coupled production of succinate, it is understood that any gene disruption that increases the production of succinate can be introduced into a host microbial organism, as desired.

[0144] Therefore, the invention provides microbial organisms having one or more genetic modifications that increase production of succinate. Such genetic modifications include, but are not limited to disruptions encode proteins or enzymes selected from pyruvate kinase, alcohol dehydrogenase; lactate dehydrogenase; acetate kinase, pyruvate oxidase, and pyruvate formate lyase. Additional metabolic modifications can be identified by an in silico method such as OptKnock, as disclosed herein. The set of metabolic modifications can include functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion. For succinate production, metabolic modifications can be selected from disruption of genes that encode proteins or enzymes selected from pyruvate kinase, alcohol dehydrogenase; lactate dehydrogenase; acetate kinase, pyruvate oxidase, pyruvate formate lyase, PTS system of glucose transport, and the like as described in the Examples.

[0145] Thus, the invention provides a non-naturally occurring microbial organism comprising one or more gene disruptions that confer increased production of succinate, in particular when such genetic modifications are combined with other microbial organisms of the invention having increased production of succinate. In one embodiment, the one or more gene disruptions do not couple growth with production of succinate. In another embodiment, the one or more gene disruptions confer growth-coupled production of succinate, and can, for example, confer stable growth-coupled production of succinate. In another embodiment, the one or more gene disruptions can confer obligatory coupling of succinate production to growth of the microbial organism. Such one or more gene disruptions reduce the activity of the respective one or more encoded enzymes.

[0146] The non-naturally occurring microbial organism can have one or more gene disruptions selected from proteins or enzymes selected from pyruvate kinase, alcohol dehydrogenase; lactate dehydrogenase; acetate kinase, pyruvate oxidase, and pyruvate formate lyase. As disclosed herein, the one or more gene disruptions can be a deletion. Such non-naturally occurring microbial organisms of the invention include bacteria, yeast, fungus, or any of a variety of other microorganisms applicable to fermentation processes, as disclosed herein.

[0147] Thus, the invention provides a non-naturally occurring microbial organism, comprising one or more gene disruptions, where the one or more gene disruptions occur in genes encoding proteins or enzymes where the one or more gene disruptions confer increased production of succinate in the organism. The production of succinate can be growth-coupled or not growth-coupled. In a particular embodiment, the production of succinate can be obligatorily coupled to growth of the organism, as disclosed herein.

[0148] As disclosed herein, the genetic alterations including gene disruptions result in increased production and an enhanced level of succinate production compared to a strain that does not contain such metabolic alterations, under appropriate culture conditions.

Appropriate conditions include, for example, those disclosed herein, including conditions such as particular carbon sources or reactant availabilities and/or adaptive evolution.

[0149] Given the teachings and guidance provided herein, those skilled in the art will understand that to introduce a metabolic alteration such as disruption of an enzymatic reaction, it is necessary to disrupt the catalytic activity of the one or more enzymes involved in the reaction. Alternatively, a metabolic alteration can include disruption of expression of a regulatory protein or cofactor necessary for enzyme activity or maximal activity. Disruption can occur by a variety of methods including, for example, deletion of an encoding gene or incorporation of a genetic alteration in one or more of the encoding gene sequences. The encoding genes targeted for disruption can be one, some, or all of the genes encoding enzymes involved in the catalytic activity. For example, where a single enzyme is involved in a targeted catalytic activity, disruption can occur by a genetic alteration that reduces or eliminates the catalytic activity of the encoded gene product. Similarly, where the single enzyme is multimeric, including heteromeric, disruption can occur by a genetic alteration that reduces or destroys the function of one or all subunits of the encoded gene products.

Destruction of activity can be accomplished by loss of the binding activity of one or more subunits required to form an active complex, by destruction of the catalytic subunit of the multimeric complex or by both. Other functions of multimeric protein association and activity also can be targeted in order to disrupt a metabolic reaction of the invention. Such other functions are well known to those skilled in the art. Similarly, a target enzyme activity can be reduced or eliminated by disrupting expression of a protein or enzyme that modifies and/or activates the target enzyme, for example, a molecule required to convert an

apoenzyme to a holoenzyme. Further, some or all of the functions of a single polypeptide or multimeric complex can be disrupted according to the invention in order to reduce or abolish the catalytic activity of one or more enzymes involved in a reaction or metabolic modification of the invention. Similarly, some or all of enzymes involved in a reaction or metabolic modification of the invention can be disrupted so long as the targeted reaction is reduced or eliminated.

[0150] Given the teachings and guidance provided herein, those skilled in the art also will understand that an enzymatic reaction can be disrupted by reducing or eliminating reactions encoded by a common gene and/or by one or more orthologs of that gene exhibiting similar or substantially the same activity. Reduction of both the common gene and all orthologs can lead to complete abolishment of any catalytic activity of a targeted reaction. However, disruption of either the common gene or one or more orthologs can lead to a reduction in the catalytic activity of the targeted reaction sufficient to promote coupling of growth to product biosynthesis. Exemplified herein are both the common genes encoding catalytic activities for a variety of metabolic modifications as well as their orthologs. Those skilled in the art will understand that disruption of some or all of the genes encoding a enzyme of a targeted metabolic reaction can be practiced in the methods of the invention and incorporated into the non-naturally occurring microbial organisms of the invention in order to achieve the increased production of succinate or growth-coupled product production.

[0151] Each of the strains can be supplemented with additional deletions if it is determined that a further increase in the production of succinate and/or coupling the formation of the product with biomass formation is desired. Alternatively, some other enzymes not known to possess significant activity under the growth conditions can become active due to adaptive evolution or random mutagenesis. Such activities can also be knocked out.

[0152] Succinate can be harvested or isolated at any time point during the culturing of the microbial organism, for example, in a continuous and/or near-continuous culture period, as disclosed herein. Generally, the longer the microorganisms are maintained in a continuous and/or near-continuous growth phase, the proportionally greater amount of succinate can be produced.

[0153] Therefore, the invention additionally provides a method for producing succinate that includes culturing a non-naturally occurring microbial organism having one or more gene disruptions, as disclosed herein. The disruptions can occur in one or more genes encoding an enzyme that increases production of succinate, including coupling succinate production to growth of the microorganism when the gene disruption reduces or eliminates an activity of the enzyme. For example, the disruptions can confer stable growth-coupled production of succinate onto the non-naturally microbial organism.

[0154] In some embodiments, the gene disruption can include a complete gene deletion. In some embodiments other methods to disrupt a gene include, for example, frameshifting by omission or addition of oligonucleotides or by mutations that render the gene inoperable. One skilled in the art will recognize the advantages of gene deletions, however, because of the stability it confers to the non-naturally occurring organism from reverting to a parental phenotype in which the gene disruption has not occurred. In particular, the gene disruptions are selected from the gene sets as disclosed herein.

[0155] Gene disruptions, including gene deletions, are introduced into host organism by methods well known in the art. A particularly useful method for gene disruption is by homologous recombination, as disclosed herein. The engineered strains can be characterized by measuring the growth rate, the substrate uptake rate, and/or the product/byproduct secretion rate. Cultures can be grown and used as inoculum for a fresh batch culture for which measurements are taken during exponential growth. The growth rate can be determined by measuring optical density using a spectrophotometer (A600). Concentrations of glucose and other organic acid byproducts in the culture supernatant can be determined by well known methods such as HPLC, GC-MS or other well known analytical methods suitable for the analysis of the desired product, as disclosed herein, and used to calculate uptake and secretion rates.

[0156] Strains containing gene disruptions can exhibit suboptimal growth rates until their metabolic networks have adjusted to their missing functionalities. To assist in this adjustment, the strains can be adaptively evolved. By subjecting the strains to adaptive evolution, cellular growth rate becomes the primary selection pressure and the mutant cells are compelled to reallocate their metabolic fluxes in order to enhance their rates of growth. This reprogramming of metabolism has been recently demonstrated for several E. coli mutants that had been adaptively evolved on various substrates to reach the growth rates predicted a priori by an in silico model (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). The growth improvements brought about by adaptive evolution can be accompanied by enhanced rates of succinate production. The strains are generally adaptively evolved in replicat, running in parallel, to account for differences in the evolutionary patterns that can be exhibited by a host organism (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Fong et al, J. Bacteriol. 185:6400-6408 (2003); Ibarra et al, Nature 420: 186-189 (2002)) that could potentially result in one strain having superior production qualities over the others.

Evolutions can be run for a period of time, typically 2-6 weeks, depending upon the rate of growth improvement attained. In general, evolutions are stopped once a stable phenotype is obtained.

[0157] Following the adaptive evolution process, the new strains are characterized again by measuring the growth rate, the substrate uptake rate, and the product/byproduct secretion rate. These results are compared to the theoretical predictions by plotting actual growth and production yields along side the production envelopes from metabolic modeling. The most successful design/evolution combinations are chosen to pursue further, and are characterized in lab-scale batch and continuous fermentations. The growth-coupled biochemical production concept behind the methods disclosed herein such as OptKnock approach should also result in the generation of genetically stable overproduces. Thus, the cultures are maintained in continuous mode for an extended period of time, for example, one month or more, to evaluate long-term stability. Periodic samples can be taken to ensure that yield and productivity are maintained.

[0158] Adaptive evolution is a powerful technique that can be used to increase growth rates of mutant or engineered microbial strains, or of wild-type strains growing under unnatural environmental conditions. It is especially useful for strains designed via methods such as OptKnock, which results in growth-coupled product formation. Therefore, evolution toward optimal growing strains will indirectly optimize production as well. Unique strains of E. coli K-12 MG1655 were created through gene knockouts and adaptive evolution. (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). In this work, all adaptive evolutionary cultures were maintained in prolonged exponential growth by serial passage of batch cultures into fresh medium before the stationary phase was reached, thus rendering growth rate as the primary selection pressure. Knockout strains were constructed and evolved on minimal medium supplemented with different carbon substrates (four for each knockout strain).

Evolution cultures were carried out in duplicate or triplicate, giving a total of 50 evolution knockout strains. The evolution cultures were maintained in exponential growth until a stable growth rate was reached. The computational predictions were accurate (within 10%) at predicting the post-evolution growth rate of the knockout strains in 38 out of the 50 cases examined. Furthermore, a combination of OptKnock design with adaptive evolution has led to improved lactic acid production strains (Fong et al, Biotechnol. Bioeng. 91 :643-648 (2005)). Similar methods can be applied to the strains disclosed herein and applied to various host strains.

[0159] There are a number of developed technologies for carrying out adaptive evolution. Exemplary methods are disclosed herein. In some embodiments, optimization of a non- naturally occurring organism of the present invention includes utilizing adaptive evolution techniques to increase succinate production and/or stability of the producing strain.

[0160] Serial culture involves repetitive transfer of a small volume of grown culture to a much larger vessel containing fresh growth medium. When the cultured organisms have grown to saturation in the new vessel, the process is repeated. This method has been used to achieve the longest demonstrations of sustained culture in the literature (Lenski and

Travisano, Proc. Natl. Acad. Sci. USA 91 :6808-6814 (1994)) in experiments which clearly demonstrated consistent improvement in reproductive rate over a period of years. Typically, transfer of cultures is usually performed during exponential phase, so each day the transfer volume is precisely calculated to maintain exponential growth through the next 24 hour period. Manual serial dilution is inexpensive and easy to parallelize.

[0161] In continuous culture the growth of cells in a chemostat represents an extreme case of dilution in which a very high fraction of the cell population remains. As a culture grows and becomes saturated, a small proportion of the grown culture is replaced with fresh media, allowing the culture to continually grow at close to its maximum population size. Chemostats have been used to demonstrate short periods of rapid improvement in

reproductive rate (Dykhuizen, Methods Enzymol. 613-631 (1993)). The potential usefulness of these devices was recognized, but traditional chemostats were unable to sustain long periods of selection for increased reproduction rate, due to the unintended selection of dilution-resistant (static) variants. These variants are able to resist dilution by adhering to the surface of the chemostat, and by doing so, outcompete less adherent individuals, including those that have higher reproductive rates, thus obviating the intended purpose of the device (Chao and Ramsdell, J. Gen. Microbiol 20: 132-138 (1985)). One possible way to overcome this drawback is the implementation of a device with two growth chambers, which periodically undergo transient phases of sterilization, as described previously (Marliere and Mutzel, U.S. Patent No. 6,686,194).

[0162] Evolugator™ is a continuous culture device developed by Evolugate, LLC (Gainesville, FL) and exhibits significant time and effort savings over traditional evolution techniques (de Crecy et al.,. Appl. Microbiol. Biotechnol. 77:489-496 (2007)). The cells are maintained in prolonged exponential growth by the serial passage of batch cultures into fresh medium before the stationary phase is attained. By automating optical density measurement and liquid handling, the Evolugator™ can perform serial transfer at high rates using large culture volumes, thus approaching the efficiency of a chemostat in evolution of cell fitness. For example, a mutant of Acinetobacter sp ADP1 deficient in a component of the translation apparatus, and having severely hampered growth, was evolved in 200 generations to 80% of the wild-type growth rate. However, in contrast to the chemostat which maintains cells in a single vessel, the machine operates by moving from one "reactor" to the next in subdivided regions of a spool of tubing, thus eliminating any selection for wall-growth. The transfer volume is adjustable, and normally set to about 50%. A drawback to this device is that it is large and costly, thus running large numbers of evolutions in parallel is not practical.

Furthermore, gas addition is not well regulated, and strict anaerobic conditions are not maintained with the current device configuration. Nevertheless, this is an alternative method to adaptively evolve a production strain.

[0163] As disclosed herein, a nucleic acid encoding a desired activity of a succinate pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a succinate pathway enzyme or protein to increase production of succinate. For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator.

[0164] One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >10 ⁴ ). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al, Biomol.Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax.

Biomol.Eng 22: 1-9 (2005).; and Sen et al, Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes.

Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (K _m ), including broadening substrate binding to include non-natural substrates; inhibition (K;), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

[0165] A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a succinate pathway enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J Theor.Biol. 234:497-509 (2005)); Error- prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al, Nucleic Acids Res. 32:el45 (2004); and Fujii et al, Nat. Protoc. 1 :2493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA 91 : 10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al, Nucleic Acids Res 26:681-683 (1998)).

[0166] Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:el8 (1999); and Volkov et al, Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates

(RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al, J. Molec. Catalysis 26: 119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules;

(Bergquist and Gibbs, Methods Mol.Biol 352: 191-204 (2007); Bergquist et al, Biomol.Eng 22:63-72 (2005); Gibbs et al, Gene 271 : 13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al, Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al, Nat. Biotechnol. 17: 1205-1209 (1999)); Thio- Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al, Nucleic Acids Res 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al, Proc. Natl. Acad. Sci. USA 98: 11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al, Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of "universal" bases such as inosine, and replication of an inosine- containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al, Biotechnol. J. 3:74-82 (2008); Wong et al, Nucleic Acids Res. 32:e26 (2004); and Wong et al, Anal. Biochem. 341 : 187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode "all genetic diversity in targets" and allows a very high diversity for the shuffled progeny (Ness et al, Nat. Biotechnol. 20: 1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al, Nucleic Acids Res. 33:el 17 (2005)).

[0167] Further methods include Sequence Homo logy-Independent Protein

Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al, Nat. Biotechnol.

19:456-460 (2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564- 586 (1991); and Reidhaar-Olson et al. Science 241 :53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al, Angew. Chem. Int. Ed Engl. 40:3589- 3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al, Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al, J. Mol. Biol. 260:359-3680 (1996)).

[0168] Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471

(2005) ); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al, Proc. Natl. Acad. Sci. USA 99: 15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al, Nat. Protoc. 2:891-903 (2007); and Reetz et al, Angew. Chem. Int. Ed Engl. 45:7745-7751

(2006) ).

[0169] Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein.

[0170] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. EXAMPLE I

Increasing Production of Succinate in an Organism

[0171] This example describes increasing production of succinate in a microbial organism.

[0172] Succinate can be produced solely from sugars such as glucose and C0 ₂ with a maximum theoretical yield of 1.71 mol succinate/mol glucose or 1.12 g/g (see Figure 1).

7 C ₆ Hi ₂ 0 ₆ + 6 C0 ₂ = 12 C ₄ H ₆ 0 ₄ + 6 H ₂ 0

[0173] Identical or similar g/g yields are achievable on other carbohydrates (for example, xylose and arabinose).

8.4 C ₅ Hio0 ₅ + 6 C0 ₂ = 12 C ₄ H ₆ 0 ₄ + 6 H ₂ 0

[0174] Alternatively, assuming that an ample source of reducing equivalents (for example, CO or H ₂ ) is present, succinate can be produced completely from CO or C0 ₂ via the rTCA cycle, associated anapleurotic reactions, and enzymes for the extraction of reducing equivalents from CO and/or H ₂ (see Figure 2).

7 CO + 3 H ₂ 0 = C ₄ H ₆ 0 ₄ + 3 C0 ₂

7 H ₂ + 4 C0 ₂ = C ₄ H ₆ 0 ₄ + 4 H ₂ 0

[0175] Yet another option is the provision of sufficient reducing equivalents to allow for the production of 2.00 mol succinate/mol glucose or 1.31 g/g (see Figure 3). Note that this yield is greater than what can be achieved on glucose alone.

C ₆ Hi ₂ 0 ₆ + 2 C0 ₂ + 2 H ₂ = 2 C ₄ H ₆ 0 ₄ + 2 H ₂ 0

[0176] Similar yield increases can be achieved using other carboxyhydrates (for example, xylose or arabinose) in combination with a source of reducing equivalents (for example, CO and/or H ₂ ).

6/5 C ₅ Hio0 ₅ + 2 C0 ₂ + 2 H ₂ = 2 C ₄ H ₆ 0 ₄ + 2 H ₂ 0

6/5 C ₅ Hio0 ₅ + 2 CO = 2 C ₄ H ₆ 0

[0177] Supplementing carbohydrate feeds with external reducing equivalents is an attractive option as it reduces the number of TCA enzymes required (see Figure 3 compared to Figure 1). For example, although reactions for the conversion of oxaloacetate or malate to succinate provide for the production of succinate at high yield, reactions associated with the conversion of oxaloacetate to alpha-ketoglutarate or pyruvate to acetyl-CoA are not required. This is particularly useful for the engineering of a eukaryotic organism, such as

Saccharomyces cerevisiae, for the production of succinate. Some eukaryotic organisms can be used advantageously over some bacterial species, for example, Escherichia coli, in that they can tolerate lower pH conditions. Production of succinate at a pH lower than the pKa's of the acid groups (pKal = 4.2, pKa2 = 5.6) is desirable since a higher percentage of final product will be in the acid form (that is,, succinic acid) and not the salt form.

[0178] A major challenge associated with engineering a eukaryotic organism to achieve a high yield of succinate from carbohydrates alone is that several requisite enzymes are not exclusively localized to the cytosol. In fact, enzymes such as pyruvate dehydrogenase and citrate synthase are predominantly mitochondrial or peroxisomal. Thus engineering a eukaryotic organism to achieve a high yield of succinate would require shuttling of metabolic precursors across cellular compartments or extensive strain engineering to localize several of the requisite enzymes to one compartment, preferably the cytosol. The embodiment described here provides a simplification of the pathway engineering by limiting the number of TCA cycle enzymes that are required to carry high flux. One can thus envision the development of a succinate producing eukaryotic microorganism in which most, if not all, of the enzymes in Figure 3 are cytosolic.

[0179] Figures 1 and 3 depict the utilization of glucose. It is well known in the art that glucose can be convereted to phosphoenolpyruvate by glycolysis. Such a conversion can include, for example, the conversion of glucose to glucose-6-phosphate by hexokinase or glucokinase. Glucose-6-phosphate can be converted to fructose-6-phosphate by glucose phosphate isomerase. Fructose-6-phosphate can be converted to fructose 1,6-diphosphate by phosphofructokinase. The conversion of fructose 1,6-diphosphate can be converted to glyceraldehyde 3 -phosphate and dihydroxyacetone phosphate by fructose diphosphate aldolase. Glyceraldehyde 3-phosphate can be converted to 3-phosphoglyceroyl phosphate by glyceraldehydephosphate dehydrogenase. The conversion of 3-phosphoglyceroyl phosphate to 3 -phosphogly cerate can be carried out by phosphoglycerate kinase. 3-Phosphoglycerate can be converted to 2-phosphoglycerate by phosphglyceromutase. The conversion of 2- phosphoglycerate to phosphoenolpyruvate can be carried out by enolase. Finally, phosphoenolpyruvate can be converted to pyruvate by pyruvate kinase.

[0180] This example describes exemplary pathways for increasing succinate production.

EXAMPLE II

Strategies for Increased Production of Succinate

[0181] This example describes exemplary strategies to increase production of succinate.

[0182] Several strain engineering strategies can be implemented to increase the production of succinate in an organism and to couple it to growth.

[0183] Overexpression of carbon- fixing enzymes such as PEP carboxylase or PEP carboxykinase (PEPCK), malic enzyme, and pyruvate carboxylase can be used to redirect carbon flux into succinate formation. When using the rTCA enzymes with C0 ₂ and H ₂ only, production of succinate can be further facilitated by the increased expression of isocitrate lyase and malate synthase, enzymes of the glyoxylate shunt. Approaches for increasing flux through the glyoxylate shunt include deletion of the repressor for the glyoxylate operon, for example, iclR in E. coli, which represses the transcription of the glyoxylate operon. Other approaches for improving carbon flux through the glyoxylate shunt include deregulating the shunt from catabolite repression and from repression under anaerobic conditions, for example, by deleting arcA. Catabolite repression can be removed or reduced by truncating the gene responsible for forming cAMP, adenylate cyclase, cyaA (Crasnier et al, Mol. Gen. Genet 243:409-416 (1994)), and by mutating the catabolite repressor protein, crp (Eppler and Boos, Mol. Microbiol. 33:1221-1231 (1999); Karimova et al, Res. Microbiol. 155:76-79 (2004); Zhu and Lin, J. Bacteriol. 170:2352-2358 (1988)). Decreasing or eliminating byproducts such as ethanol, acetate, lactate and formate can be used to improve yields of succinate. In E. coli and other prokaryotes, decreasing or eliminating such byproducts can be effected by deletions in alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), acetate kinase (ackA), pyruvate oxidase (poxB), and pyruvate formate lyase (pflB). The homologue of pflB, pyruvate formate-lyase 2-ketobutyrate formate-lyase (tdcE), can also be deleted in E. coli. Further, deletion of transporters such as the phosphotransferase system (PTS) of glucose uptake increases the PEP pool in the organism and this has been demonstrated to improve succinate production in the literature. This can be accomplished by deletion, mutataion or truncation of ptsG, ptsH, ptsl or err or their combinations (Zhang et al, Proc. Natl. Acad. Sci. USA 106(48):20180-20185 (2009), Flores et al, Mol. Microbiol. Biotechnol. 13: 105-116 (2007); Sanchez et al, Biotechnol. Prog. 21(2):358-365 (2005)). This deletion can optionally be accompanied by overexpression of glucokinase encoded by glk and galactose permease encoded by galP. Similarly, deletion in pyruvate kinase (pykA, pykF) prevents the conversion of PEP to pyruvate and improves succinate production. Further, high concentrations of C0 ₂ in the fermenters allow the function of PEPCK and pyruvate carboxylase in the anaplerotic direction, as needed for succinate production. While exemplified above with specific genes, it is understood by those skilled in the art that genes performing the same or similar functions can be genetically modified in the appropriate host organism to achieve a similar improvement in succinate production.

[0184] Similar strategies to those proposed above can be used for production of succinate in yeasts such as Saccharomyces cerevisiae and Candida. In yeast, succinate production can be improved by elimination or reduction of byproducts such as glycerol and ethanol by deleting or attenuating g3pdl, g3pd2, pdcl, pdc5 and pdc6. In addition, respiration can be reduced by attenuating or deleting ndel , nde2, and ndil . Redox availability for production of succinate can be enhanced by reducing or deleting the glycerol-3 -phosphate shuttle encoded by gut2m. Reduced activity of pyruvate kinase encoded by pykl and pyk2 can also be used to increase the pool of PEP for succinate production.

[0185] Following are some gene candidates for isocitrate lyase:

[0186] Following are some gene candidates for malate synthase:

Protein GenBank ID GI Number Organism

aceB NP_418438.1 16131840 Escherichia coli

MLS1 NP_014282.1 6324212 Saccharomyces cerevisiae glcB CAPO 1154.1 169152249 Acinetobacter baumannii

PA13 4357 EGM14762.1 334835919 Pseudomonas aeruginosa [0187] This example describes increased production of succinate.

EXAMPLE III

Increased Production of Succinate in Microbial Organisms

[0188] This example describes engineering of microbial organisms for increased production of succinate.

[0189] In E. coli, the relevant genes are expressed in a synthetic operon behind an inducible promoter on a medium- or high-copy plasmid; for example the PBAD promoter which is induced by arabinose, on a plasmid of the pBAD series (Guzman et al, J. Bacteriol. 177:4121-4130 (1995)). In S. cerevisiae, genes are integrated into the chromosome behind the PDCl promoter, replacing the native pyruvate carboxylase gene. It has been reported that this results in higher expression of foreign genes than from a plasmid (Ishida et al, Appl. Environ. Microbiol. 71 : 1964-1970 (2005)), and will also ensure expression during anaerobic conditions.

[0190] Cells containing the relevant constructs are grown in suitable growth media, for example, minimal media containing glucose. The addition of arabinose can be included in the case of E. coli containing genes expressed under the PBAD promoter. Periodic samples are taken for both gene expression and enzyme activity analysis. Enzyme activity assays are performed on crude cell extracts using procedures well known in the art. Alternatively, assays based on the oxidation of NAD(P)H, which is produced in all dehydrogenase reaction steps and detectable by spectrophotometry can be utilized. In addition, antibodies can be used to detect the level of particular enzymes. In lieu of or in addition to enzyme activity measurements, R A can be isolated from parallel samples and transcript of the gene of interest measured by reverse transcriptase PCR. Any constructs lacking detectable transcript expression are reanalyzed to ensure the encoding nucleic acids are harbored in an expressible form. Where transcripts are detected, this result indicates either a lack of translation or production of inactive enzyme. A variety of methods well known in the art can additionally be employed, such as codon optimization, engineering a strong ribosome binding site, use of a gene from a different species, and prevention of N-glycosylation (for expression of bacterial enzymes in yeast) by conversion of Asn residues to Asp. Once all required enzyme activities are detected, the next step is to measure the production of succinate in vivo. Triplicate shake flask cultures are grown aerobically, anaerobically or microaerobically, depending on the conditions required, and periodic samples taken. Organic acids present in the culture supematants are analyzed by HPLC using the Aminex AH-87X column. The elution time of succinate will be determined using a standard purchased from a chemical supplier.

EXAMPLE IV

Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting Reducing Equivalents from Syngas and Exemplary Reductive TCA Cycle Enzymes

[0191] Enzymes of the reductive TCA cycle useful in the non-naturally occurring microbial organisms of the present invention include one or more of ATP-citrate lyase and three C0 ₂ -fixing enzymes: isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate :ferredoxin oxidoreductase. The presence of ATP-citrate lyase or citrate lyase and alpha-ketoglutarate:ferredoxin oxidoreductase indicates the presence of an active reductive TCA cycle in an organism. Enzymes for each step of the reductive TCA cycle are shown below.

[0192] ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase, catalyzes the ATP-dependent cleavage of citrate to oxaloacetate and acetyl-CoA. ACL is an enzyme of the RTCA cycle that has been studied in green sulfur bacteria Chlorobium limicola and Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was cloned and characterized in E. coli (Kanao et al, Eur. J. Biochem. 269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, is irreversible and activity of the enzyme is regulated by the ratio of ADP/ATP. A recombinant ACL from Chlorobium tepidum was also expressed in E. coli and the holoenzyme was reconstituted in vitro, in a study elucidating the role of the alpha and beta subunits in the catalytic mechanism (Kim and Tabita, J. Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been identified in Balnearium lithotrophicum, Sulfurihydrogenibium subterraneum and other members of the bacterial phylum Aquificae (Hugler et al, Environ. Microbiol. 9:81-92 (2007)). This acitivy has been reported in some fungi as well. Exemplary organisms include Sordaria macrospora (Nowrousian et al., Curr. Genet. 37:189-93 (2000), Aspergillus nidulans , Yarrowia lipolytica (Hynes and Murray, Eukaryotic Cell, July: 1039-1048, (2010) and Aspergillus niger (Meijer et al. J. Ind. Microbiol. Biotechnol. 36:1275-1280 (2009). Other candidates can be found based on sequence homology. Information related to these enzymes is tabulated below: Protein GenBank ID GI Number Organism aclA BAB21376.1 12407237 Chlorobium limicola

aclB BAB21375.1 12407235 Chlorobium limicola

aclA AAM72321.1 21647054 Chlorobium tepidum

aclB AAM72322.1 21647055 Chlorobium tepidum

aclA ABI50076.1 114054981 Balnearium lithotrophicum

aclB ABI50075A 114054980 Balnearium lithotrophicum

aclA ABI50085.1 114055040 Sulfurihydrogenibium subterraneum aclB ABI50084.1 114055039 Sulfurihydrogenibium subterraneum aclA AAX76834.1 62199504 Sulfurimonas denitrificans

aclB AAX76835.1 62199506 Sulfurimonas denitrificans

acll XP_504787.1 50554757 Yarrowia lipolytica

acl2 XP 503231.1 50551515 Yarrowia lipolytica

SPBC1703.07 NP_596202.1 19112994 Schizosaccharomyces pombe

SPAC22A12.16 NP_593246.1 19114158 Schizosaccharomyces pombe acll CAB76165.1 7160185 Sordaria macrospora

acl2 CAB76164.1 7160184 Sordaria macrospora

aclA CBF86850.1 259487849 Aspergillus nidulans

aclB CBF86848 259487848 Aspergillus nidulans

[0193] In some organisms the conversion of citrate to oxaloacetate and acetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzed by two separate enzymes, citryl- CoA synthetase (EC 6.2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl.

Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes the activation of citrate to citryl-CoA. The Hydrogenobacter thermophilus enzyme is composed of large and small subunits encoded by ccsA and ccsB, respectively (Aoshima et al, Mol. Micrbiol.

52:751-761 (2004)). The citryl-CoA synthetase of Aquifex aeolicus is composed of alpha and beta subunits encoded by sucCl and sucDl (Hugler et al, Environ. Microbiol. 9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and acetyl-CoA. This enzyme is a homotrimer encoded by ccl in Hydrogenobacter thermophilus (Aoshima et al, Mol. Microbiol. 52:763-770 (2004)) and aq_150 in Aquifex aeolicus (Hugler et al, supra (2007)). The genes for this mechanism of converting citrate to oxaloacetate and citryl-CoA have also been reported recently in Chlorobium tepidum (Eisen et al, PNAS 99(14): 9509-14 (2002). Protein GenBank ID GI Number Organism ccsA BAD 17844.1 46849514 Hydrogenobacter thermophilus ccsB BAD 17846.1 46849517 Hydrogenobacter thermophilus sucCl AAC07285 2983723 Aquifex aeolicus

sucDl AAC07686 2984152 Aquifex aeolicus

ccl BAD17841.1 46849510 Hydrogenobacter thermophilus aq_150 AAC06486 2982866 Aquifex aeolicus

CT0380 NP_661284 21673219 Chlorobium tepidum

CT0269 NP_661173.1 21673108 Chlorobium tepidum

CT1834 AAM73055.1 21647851 Chlorobium tepidum

[0194] Oxaloacetate is converted into malate by malate dehydrogenase (EC 1.1.1.37), an enzyme which functions in both the forward and reverse direction. S. cerevisiae possesses three copies of malate dehydrogenase, MDHl (McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11 :370-380 (1991); Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively. E. coli is known to have an active malate dehydrogenase encoded by mdh.

[0195] Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygen availability. FumB is oxygen sensitive and is active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is active under aerobic growth conditions (Tseng et al, J. Bacteriol. 183:461-467

(2001);Woods et al, Biochim. Biophys. Acta 954: 14-26 (1988); Guest et al, J. Gen.

Microbiol. 131 :2971-2984 (1985)). S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass et al, J. Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes are found in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell. Biol. 31 :961-975 (1999)), Thermus thermophilus (Mizobata et al, Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al, J. Biochem. 89: 1923-1931 (1981)). Similar enzymes with high sequence homology include fuml from Arabidopsis thaliana and fumC from

Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum

thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).

[0196] Fumarate reductase catalyzes the reduction of fumarate to succinate. The fumarate reductase of E. coli, composed of four subunits encoded by frdABCD, is membrane- bound and active under anaerobic conditions. The electron donor for this reaction is menaquinone and the two protons produced in this reaction do not contribute to the proton gradient (Iverson et al, Science 284: 1961-1966 (1999)). The yeast genome encodes two soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto et al, DNA Res. 3:263- 267 (1996)) and FRDS2 (Muratsubaki et al, Arch. Biochem. Biophys. 352: 175-181 (1998)), which localize to the cytosol and promitochondrion, respectively, and are used during anaerobic growth on glucose (Arikawa et al., FEMS Microbiol. Lett. 165: 111-116 (1998)).

Protein GenBank ID GI Number Organism

FRDS1 P32614 418423 Saccharomyces cerevisiae

FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdA NP_418578.1 16131979 Escherichia coli

frdB NP_418577.1 16131978 Escherichia coli Protein GenBank ID GI Number Organism frdC NP_418576.1 16131977 Escherichia coli

frdD NP_418475.1 16131877 Escherichia coli

[0197] The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5). The product of the LSC1 and LSC2 genes of S.

cerevisiae and the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below:

[0198] Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also known as 2- oxoglutarate synthase or 2-oxoglutarate:ferredoxin oxidoreductase (OFOR), forms alpha- ketoglutarate from C02 and succinyl-CoA with concurrent consumption of two reduced ferredoxin equivalents. OFOR and pyruvate :ferredoxin oxidoreductase (PFOR) are members of a diverse family of 2-oxoacid:ferredoxin (flavodoxin) oxidoreductases which utilize thiamine pyrophosphate, CoA and iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and FAD as electron carriers (Adams et al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in this class are reversible and function in the carboxylation direction in organisms that fix carbon by the RTCA cycle such as Hydrogenobacter thermophilus, Desulfobacter hydrogenophilus and Chlorobium species (Shiba et al. 1985; Evans et al, Proc. Natl. Acad. Sci. U.S.A. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme from H. thermophilus, encoded by korAB, has been cloned and expressed in E. coli (Yun et al, Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five subunit OFOR from the same organism with strict substrate specificity for succinyl-CoA, encoded by forDABGE, was recently identified and expressed in E. coli (Yun et al, Biochem. Biophys. Res. Commun. 292:280-286 (2002)). The kinetics of C02 fixation of both H. thermophilus OFOR enzymes have been characterized (Yamamoto et al., Extremophiles 14:79-85 (2010)). A C0 ₂ -fixing OFOR from Chlorobium thiosulfatophilum has been purified and characterized but the genes encoding this enzyme have not been identified to date. Enzyme candidates in Chlorobium species can be inferred by sequence similarity to the H. thermophilus genes. For example, the Chlorobium limicola genome encodes two similar proteins. Acetogenic bacteria such as Moorella thermoacetica are predicted to encode two OFOR enzymes. The enzyme encoded by Moth_0034 is predicted to function in the C02-assimilating direction. The genes associated with this enzyme, Moth_0034 have not been experimentally validated to date but can be inferred by sequence similarity to known OFOR enzymes.

[0199] OFOR enzymes that function in the decarboxylation direction under physiological conditions can also catalyze the reverse reaction. The OFOR from the thermoacidophilic archaeon Sulfolobus sp. strain 7, encoded by ST2300, has been extensively studied (Zhang et al, supra, 1996). A plasmid-based expression system has been developed for efficiently expressing this protein in E. coli (Fukuda et al, Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). The OFOR encoded by Ape 1472 /Ape 1473 from

Aeropyrum pernix str. Kl was recently cloned into E. coli, characterized, and found to react with 2-oxoglutarate and a broad range of 2-oxoacids (Nishizawa et al, FEBS Lett. 579:2319- 2322 (2005)). Another exemplary OFOR is encoded by oorDABC in Helicobacter pylori (Hughes et al, J. Bacteriol. 180:1119-1128 (1998)). An enzyme specific to alpha- ketoglutarate has been reported in Thauera aromatica (Dorner and Boll, J. Bacteriol. 184 (14), 3975-83 (2002)). A similar enzyme can be found in Rhodospirillum rubrum by sequence homology. A two subunit enzyme has also been identified in Chlorobium tepidum (Eisen et al, Proc. Natl. Acad. Sci. USA 99(14):9509-9514 (2002)).

Protein GenBank ID GI Number Organism

korA BAB21494 12583691 Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacter thermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forA BAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.1 14970996 Hydrogenobacter thermophilus forG BAB62135.1 14970997 Hydrogenobacter thermophilus forE BAB62136.1 14970998 Hydrogenobacter thermophilus

Clim_0204 ACD89303.1 189339900 Chlorobium limicola

Clim_0205 ACD89302.1 189339899 Chlorobium limicola Clim 1123 ACD90192.1 189340789 Chlorobium limicola

Clim_1124 ACD90193.1 189340790 Chlorobium limicola

Moth_1984 YP_430825.1 83590816 Moorella thermoacetica

Moth_1985 YP 430826.1 83590817 Moorella thermoacetica

Moth_0034 YP_428917.1 83588908 Moorella thermoacetica

ST2300 NP_378302.1 15922633 Sulfolobus sp. strain 7

Apel472 BAA80470.1 5105156 Aeropyrum pernix

Apel473 BAA80471.2 116062794 Aeropyrum pernix

oorD NP_207383.1 15645213 Helicobacter pylori

(AAC38210.1) (2935178)

oorA NP_207384.1 15645214 Helicobacter pylori

(AAC38211.1) (2935179)

oorB NP_207385.1 15645215 Helicobacter pylori

(AAC38212.1) (2935180)

oorC NP_207386.1 15645216 Helicobacter pylori

(AAC38213.1) (2935181)

CT0163 NP 661069.1 21673004 Chlorobium tepidum

CT0162 NP 661068.1 21673003 Chlorobium tepidum

korA CAA12243.2 19571179 Thauera aromatica

korB CAD27440.1 19571178 Thauera aromatica

Rru_A2721 YP_427805.1 83594053 Rhodospirillum rubrum

Rru_A2722 YP_427806.1 83594054 Rhodospirillum rubrum

[0200] Isocitrate dehydrogenase catalyzes the reversible decarboxylation of isocitrate to 2-oxoglutarate coupled to the reduction of NAD(P) ⁺ . IDH enzymes in Saccharomyces cerevisiae and Escherichia coli are encoded by IDPl and icd, respectively (Haselbeck and McAlister-Henn, J. Biol. Chem. 266:2339-2345 (1991); Nimmo, Biochem. J. 234:317-2332 (1986)). The reverse reaction in the reductive TCA cycle, the reductive carboxylation of 2- oxoglutarate to isocitrate, is favored by the NADPH-dependent C0 ₂ -fixing IDH from

Chlorobium limicola and was functionally expressed in E. coli (Kanao et al., Eur. J. Biochem. 269: 1926-1931 (2002)). A similar enzyme with 95% sequence identity is found in the C. tepidum genome in addititon to some other candidates listed below. Protein GenBank ID GI Number Organism led ACI84720.1 209772816 Escherichia coli

IDP1 AAA34703.1 171749 Saccharomyces cerevisiae

Idh BAC00856.1 21396513 Chlorobium limicola

led AAM71597.1 21646271 Chlorobium tepidum

icd NP_952516.1 39996565 Geobacter sulfurreducens icd YP 393560. 78777245 Sulfurimonas denitrificans

[0201] In H. thermophilus the reductive carboxylation of 2-oxoglutarate to isocitrate is catalyzed by two enzymes: 2-oxoglutarate carboxylase and oxalosuccinate reductase. 2- Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes the ATP-dependent carboxylation of alpha- ketoglutarate to oxalosuccinate (Aoshima and Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a large complex composed of two subunits. Biotinylation of the large (A) subunit is required for enzyme function (Aoshima et al, Mol. Microbiol. 51 :791-798 (2004)). Oxalosuccinate reductase (EC 1.1.1.-) catalyzes the NAD-dependent conversion of oxalosuccinate to D-t/zreo-isocitrate. The enzyme is a homodimer encoded by icd in H. thermophilus. The kinetic parameters of this enzyme indicate that the enzyme only operates in the reductive carboxylation direction in vivo, in contrast to isocitrate dehydrogenase enzymes in other organisms (Aoshima and Igarashi, J. Bacteriol. 190:2050-2055 (2008)). Based on sequence homology, gene candidates have also been found in Thiobacillus denitrificans and Thermocrinis albus.

Protein GenBank ID GI Number Organism

cfiA BAF34932.1 116234991 Hydrogenobacter thermophilus ci B BAF34931.1 116234990 Hydrogenobacter thermophilus led BAD02487.1 38602676 Hydrogenobacter thermophilus

Tbd_1556 YP_315314 74317574 Thiobacillus denitrificans

Tbd_1555 YP_315313 74317573 Thiobacillus denitrificans

Tbd_0854 YP 314612 74316872 Thiobacillus denitrificans

Thal_0268 YP 003473030 289548042 Thermocrinis albus

Thal_0267 YP 003473029 289548041 Thermocrinis albus

Thal_0646 YP 003473406 289548418 Thermocrinis albus [0202] Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible isomerization of citrate and iso-citrate via the intermediate czs-aconitate. Two aconitase enzymes are encoded in the E. coli genome by acnA and acnB. AcnB is the main catabolic enzyme, while AcnA is more stable and appears to be active under conditions of oxidative or acid stress (Cunningham et al, Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of aconitase in Salmonella typhimurium are encoded by acnA and acnB (Horswill and Escalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiae aconitase, encoded by ACOl, is localized to the mitochondria where it participates in the TCA cycle (Gangloff et al, Mol. Cell. Biol. 10:3551-3561 (1990)) and the cytosol where it participates in the glyoxylate shunt (Regev-Rudzki et al., Mol. Biol. Cell. 16:4163-4171 (2005)).

[0203] Pyruvate :ferredoxin oxidoreductase (PFOR) catalyzes the reversible oxidation of pyruvate to form acetyl-CoA. The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al, J. Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. Two cysteine residues in this enzyme form a disulfide bond that prtotects it against inactivation in the form of oxygen. This disulfide bond and the stability in the presence of oxygen has been found in other Desulfovibrio species also (Vita et al, Biochemistry, 47: 957-64 (2008)). The M. thermoacetica PFOR is also well characterized (Menon and Ragsdale, Biochemistry 36:8484- 8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, encoding a protein that is 51% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al, Eur. J. Biochem. 123:563-569 (1982)). PFORs have also been described in other organisms, including Rhodobacter capsulatas (Yakunin and Hallenbeck, Biochimica et Biophysica Acta 1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisen et al, Proc. Natl. Acad. Sci. USA 99(14): 9509-14 (2002)). The five subunit PFOR from H. thermophilus, encoded by porEDABG, was cloned into E. coli and shown to function in both the decarboxylating and C0 ₂ -assimilating directions (Ikeda et al., Biochem. Biophys. Res. Commun. 340:76-82 (2006); Yamamoto et al, Extremophiles 14:79-85 (2010)). Homologs also exist in C. carboxidivorans P7. Several additional PFOR enzymes are described in the following review (Ragsdale, S.W., Chem. Rev. 103:2333-2346 (2003)). Finally, flavodoxin reductases (e.g.,fqrB from Helicobacter pylori or Campylobacter jejuni) (St Maurice et al, J. Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al, Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below.

Protein GenBank ID GI Number Organism

DesfrDRAFT O 121 ZP 07331646.1 303245362 Desulfovibrio fructosovorans JJ

Por CAA70873.1 1770208 Desulfovibrio africanus por YP 012236.1 46581428 Desulfovibrio vulgaris str.

Hildenborough

Dde_3237 ABB40031.1 78220682 DesulfoVibrio desulfuricans

G20

Ddes_0298 YP 002478891.1 220903579 Desulfovibrio desulfuricans

subsp. desulfuricans str. ATCC 27774

Por YP_428946.1 83588937 Moorella thermoacetica

YdbK NP_415896.1 16129339 Escherichia coli nifj (CT1628) NP_662511.1 21674446 Chlorobium tepidum

CJE1649 YP 179630.1 57238499 Campylobacter jejuni nifj ADE85473.1 294476085 Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacter thermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus porA BAA95605.1 7768914 Hydrogenobacter thermophilus porB BAA95606.1 776891 Hydrogenobacter thermophilus porG BAA95607.1 7768916 Hydrogenobacter thermophilus

FqrB YP 001482096.1 157414840 Campylobacter jejuni

15645778

HP1164 NP_207955.1 Helicobacter pylori

RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

RnfE EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

Rnffi EDK33311.1 146346775 Clostridium kluyveri

[0204] The conversion of pyruvate into acetyl-CoA can be catalyzed by several other enzymes or their combinations thereof. For example, pyruvate dehydrogenase can transform pyruvate into acetyl-CoA with the concomitant reduction of a molecule of NAD into NADH. It is a multi-enzyme complex that catalyzes a series of partial reactions which results in acylating oxidative decarboxylation of pyruvate. The enzyme comprises of three subunits: the pyruvate decarboxylase (El), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This enzyme is naturally present in several organisms, including E. coli and S. cerevisiae. In the E. coli enzyme, specific residues in the El component are responsible for substrate specificity (Bisswanger, J. Biol. Chem. 256:815-82 (1981); Bremer, Eur. J. Biochem. 8:535-540 (1969); Gong et al, J. Biol. Chem. 275:13645-13653 (2000)). Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al, J. Bacteriol. 190:3851-3858 (2008); Kim et al, Appl. Environ.

Microbiol. 73: 1766-1771 (2007); Zhou et al, Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al, J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (5). Crystal structures of the enzyme complex from bovine kidney (18) and the E2 catalytic domain from Azotobacter vinelandii are available (4). Yet another enzyme that can catalyze this conversion is pyruvate formate lyase. This enzyme catalyzes the conversion of pyruvate and CoA into acetyl-CoA and formate. Pyruvate formate lyase is a common enzyme in prokaryotic organisms that is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in Escherichia coli encoded by pflB (Knappe and Sawers,

FEMS.Microbiol. Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen et al, Appl.

Microbiol. Biotechnol. 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al, Oral.Microbiol Immunol. 18:293-297 (2003)). E. coli possesses an additional pyruvate formate lyase, encoded by tdcE, that catalyzes the conversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively (Hesslinger et al, Mol. Microbiol. 27:477-492 (1998)). Both pflB and tdcE from E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by pflA. Further, a short protein encoded by yfiD in E. coli can associate with and restore activity to oxygen- cleaved pyruvate formate lyase (Vey et al., Proc.Natl. Acad. Sci. U.S.A. 105: 16137-16141 (2008). Note that pflA and pflB from E. coli were expressed in S. cerevisiae as a means to increase cytosolic acetyl-CoA for butanol production as described in WO/2008/080124. Additional pyruvate formate lyase and activating enzyme candidates, encoded by pfl and act, respectively, are found in Clostridium pasteurianum (Weidner et al, J. Bacteriol. 178:2440-2444 (1996)).

[0205] Further, different enzymes can be used in combination to convert pyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA is obtained in the cytosol by first decarboxylating pyruvate to form acetaldehyde; the latter is oxidized to acetate by

acetaldehyde dehydrogenase and subsequently activated to form acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme in several other organisms including E. coli (Kumari et al, J. Bacteriol. 177:2878-2886 (1995)), Salmonella enterica (Starai et al, Microbiology 151 :3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205 (2005)), and Moorella thermoacetica (described already). Alternatively, acetate can be activated to form acetyl-CoA by acetate kinase and phosphotransacetylase. Acetate kinase first converts acetate into acetyl-phosphate with the accompanying use of an ATP molecule. Acetyl- phosphate and CoA are next converted into acetyl-CoA with the release of one phosphate by phosphotransacetylase. Both acetate kinase and phosphotransacetlyase are well-studied enzymes in several Clostridia and Methanosarcina thermophila.

[0206] Yet another way of converting pyruvate to acetyl-CoA is via pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate, using ubiquione as the electron acceptor. In E. coli, this activity is encoded by poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiae and Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor (Koland and Gennis, Biochemistry 21 :4438-4442 (1982)); O'Brien et al., Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor. Acetate can then be converted into acetyl-CoA by either acetyl-CoA synthetase or by acetate kinase and phosphotransacetylase, as described earlier. Some of these enzymes can also catalyze the reverse reaction from acetyl-CoA to pyruvate.

[0207] For enzymes that use reducing equivalents in the form of NADH or NADPH, these reduced carriers can be generated by transferring electrons from reduced ferredoxin. Two enzymes catalyze the reversible transfer of electrons from reduced ferredoxins to NAD(P) ⁺ , ferredoxin:NAD ⁺ oxidoreductase (EC 1.18.1.3) and ferredoxin :NADP ⁺

oxidoreductase (FNR, EC 1.18.1.2). Ferredoxin:NADP ⁺ oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low-potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al, Eur. J. Biochem. 123:563-569 (1982); Fujii et al, 1977). The Helicobacter pylori FNR, encoded by HP 1164 (fqrB), is coupled to the activity of pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St. Maurice et al., J Bacteriol. 189(13):4764-4773 (2007)). An analogous enzyme is found in Campylobacter jejuni (St. Maurice et al, supra, 2007). A ferredoxin :NADP ⁺ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al, J Bacteriol. 175: 1590-1595 (1993)). Ferredoxin:NAD ⁺ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD ⁺ . In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD ⁺ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al., J. Bacteriol. 180:2915-2923 (1998)).

NADH: ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus strain TK-6, although a gene with this activity has not yet been indicated (Yoon et al. 2006). NADP oxidoreductase of C. kluyveri, encoded by nfnAB, catalyzes the concomitant reduction of ferredoxin and NAD+ with two equivalents of NADPH (Wang et al, J Bacteriol 192: 5115-5123 (2010)). Finally, the energy-conserving membrane-associated Rnf-type proteins (Seedorf et al, Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008);

Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin. Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridium carboxydivorans P7 and Clostridium ljungdahli.

Protein GenBank ID GI Number Organism

HP 1164 NP_207955.1 15645778 Helicobacter pylori

RPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris fpr BAH29712.1 225320633 Hydrogenobacter thermophilus yumC NP 391091.2 255767736 Bacillus subtilis

CJE0663 AAW35824.1 57167045 Campylobacter jejuni fpr P28861.4 399486 Escherichia coli

hcaD AAC75595.1 1788892 Escherichia coli

LOCI 00282643 NP 001149023.1 226497434 Zea mays

NfnA YP 001393861.1 153953096 Clostridium kluyveri

NfnB YP 001393862.1 153953097 Clostridium kluyveri

RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

RnfE EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

Rn B EDK33311.1 146346775 Clostridium kluyveri

CcarbDRAFT 2639 ZP 05392639.1 255525707 Clostridium carboxidivorans P7

CcarbDRAFT 2638 ZP 05392638.1 255525706 Clostridium carboxidivorans P7

CcarbDRAFT 2636 ZP 05392636.1 255525704 Clostridium carboxidivorans P7

CcarbDRAFT 5060 ZP 05395060.1 255528241 Clostridium carboxidivorans P7

CcarbDRAFT 2450 ZP 05392450.1 255525514 Clostridium carboxidivorans P7

CcarbDRAFT 1084 ZP 05391084.1 255524124 Clostridium carboxidivorans P7

CUU cll410 ADK14209.1 300434442 Clostridium ljungdahli

(RnfB)

CUU cll400 ADK14208.1 300434441 Clostridium ljungdahli

(RnfA)

CUU cll390 ADK14207.1 300434440 Clostridium ljungdahli

(RnfE)

CUU cll380 ADK14206.1 300434439 Clostridium ljungdahli

(RnfG) CUU cll370 ADK14205.1 300434438 Clostridium ljungdahli

(RnfD)

CUU cll360 ADK14204.1 300434437 Clostridium ljungdahli

(RnfC)

[0208] Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP ⁺ oxidoreductase, pyruvate :ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate :ferredoxin oxidoreductase (OFOR). The H. thermophilus gene fdxl encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al, JBiochem Mol Biol. 39:46-54 (2006)). While the gene associated with this protein has not been fully sequenced, the N- terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius . The E. coli genome encodes a soluble ferredoxin of unknown physiological function, fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, J Biochem. 126:917-926 (1999)). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al., J Bacteriol. 185:2927-2935 (2003)) and Campylobacter jejuni (van Vliet et al, FEMS Microbiol Lett. 196: 189-193 (2001)). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochem. Biophys. Res. Communications, 192(3): 1115-1122 (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7, Clostridium ljungdahli and Rhodo spirillum rubrum are predicted to encode several ferredoxins, listed in the table below.

Protein ( ,cn Bank ID GI Number Organism

fdxl BAE02673.1 68163284 Hydrogenobacter

thermophilus

M11214.1 AAA83524.1 144806 Clostridium pasteurianum

Zfx AAY79867.1 68566938 Sulfolobus acidocalarius

Fdx AAC75578.1 1788874 Escherichia coli

hp_0277 AAD07340.1 2313367 Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuni

Moth_0061 ABC 18400.1 83571848 Moorella thermoacetica Moth_1200 ABC19514.1 83572962 Moorella thermoacetica

Moth_1888 ABC20188.1 83573636 Moorella thermoacetica

Moth _2112 ABC20404.1 83573852 Moorella thermoacetica

Moth_1037 ABC19351.1 83572799 Moorella thermoacetica

CcarbDRAFT 4 ZP 05394383.1 255527515 Clostridium

383 carboxidivorans P7

CcarbDRAFT 2 ZP 05392958.1 255526034 Clostridium

958 carboxidivorans P7

CcarbDRAFT 2 ZP 05392281.1 255525342 Clostridium

281 carboxidivorans P7

CcarbDRAFT 5 ZP 05395295.1 255528511 Clostridium

296 carboxidivorans P7

CcarbDRAFT 1 ZP 05391615.1 255524662 Clostridium

615 carboxidivorans P7

CcarbDRAFT 1 ZP 05391304.1 255524347 Clostridium

304 carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermus

hydrogenoformans fdxN CAA35699.1 46143 Rhodobacter capsulatus

Rru_A2264 ABC23064.1 83576513 Rhodospirillum rubrum

Rru_A1916 ABC22716.1 83576165 Rhodospirillum rubrum

Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooF AAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605 Rhodospirillum rubrum

Alvin_2884 ADC63789.1 288897953 Allochromatium vinosum

DSM 180

fdx YP 002801146.1 226946073 Azotobacter vinelandii DJ

CKL 3790 YP 001397146.1 153956381 Clostridium kluyveri DSM

555

ferl NP 949965.1 39937689 Rhodopseudomonas palustris CGA009 fdx CAA12251.1 3724172 Thauera aromatica

CHY 2405 YP 361202.1 78044690 Carboxydothermus

hydrogenoformans fer YP 359966.1 78045103 Carboxydothermus

hydrogenoformans fer AAC83945.1 1146198 Bacillus subtilis fdxl NP_249053.1 15595559 Pseudomonas aeruginosa

PA01

yfriL AP 003148.1 89109368 Escherichia coli K-12

CLJU_c00930 ADK13195.1 300433428 Clostridium ljungdahli

CLJU cOOOlO ADK13115.1 300433348 Clostridium ljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahli

CLJU_c 17980 ADK14861.1 300435094 Clostridium ljungdahli

CLJU_c 17970 ADK14860.1 300435093 Clostridium ljungdahli

CLJU_c22510 ADK15311.1 300435544 Clostridium ljungdahli

CLJU_c26680 ADK15726.1 300435959 Clostridium ljungdahli

CLJU_c29400 ADK15988.1 300436221 Clostridium ljungdahli

[0209] Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3- mercaptopropionate, propionate, vinylacetate, and butyrate, among others.

[0210] The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product of catl of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in Trichomonas vaginalis (van Grinsven et al, J Biol Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al, J. Biol. Chem. 279(44):45337-45346 (2004)). The succinyl-CoA:acetate CoA-transferase from Acetobacter aceti, encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al., J. Bacteriol. 190(14):4933- 4940 (2008)). Similar succinyl-CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., supra, 2008), Trypanosoma brucei (Riviere et al., supra, 2004) and Clostridium kluyveri (Sohling and Gottschalk, supra, 1996). The beta- ketoadipate:succinyl-CoA transferase encoded by peal and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al., J. Bacteriol. 184(1):207-215 (2002)). The aforementioned proteins are identified below.

Protein GenBank ID GI Number Organism

catl P38946.1 729048 Clostridium kluyveri

TVAG 95550 XP 001330176 123975034 Trichomonas vaginalis G3

Tbll.02.0290 XP_828352 71754875 Trypanosoma brucei

peal AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putida aarC ACD85596.1 189233555 Acetobacter aceti

[0211] An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al, J. Biol. Chem. 272(41):25659-25667 (1997)), Bacillus subtilis, and Homo sapiens (Fukao et al., Genomics 68(2): 144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8(1): 16-23 (2002)). The aforementioned proteins are identified below.

[0212] Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoA transferase requires the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl- CoA to a 3-ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl- CoA can be catalyzed by an acetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl- CoA:acetate:CoA transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa. Exemplary enzymes include the gene products of atoAD from E. coli (Hanai et al, Appl Environ Microbiol 73:7814-7818 (2007), ctfAB from C.

acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77: 1219-1224 (2008), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al, Biosci.Biotechnol Biochem. 71 :58-68 (2007)) are shown below.

Protein GenBank ID GI Number Organism

A to A NP_416726.1 2492994 Escherichia coli

AtoD NP_416725.1 2492990 Escherichia coli

CtfA NP_149326.1 15004866 Clostridium acetobutylicum

Ct B NP_149327.1 15004867 Clostridium acetobutylicum Protein GenBank ID GI Number Organism

CtfA AAP42564.1 Clostridium

31075384 saccharoperbutylacetonicum

Ct B AAP42565.1 Clostridium

31075385 saccharoperbutylacetonicum

[0213] Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R)- Benzylsuccinate CoA-Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)). Homologs can be found in Azoarcus sp. T, Aromatoleum aromaticum EbNl, and Geobacter metallireducens GS-15. The aforementioned proteins are identified below.

[0214] Additionally, ygfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al, Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacter youngae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. The aforementioned proteins are identified below.

[0215] Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting in the cleavage of citrate to acetate and oxaloacetate. The enzyme is active under anaerobic conditions and is composed of three subunits: an acyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and a acyl lyase (beta). Enzyme activation uses covalent binding and acetylation of an unusual prosthetic group, 2'-(5"-phosphoribosyl)-3'-dephospho-CoA, which is similar in structure to acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyase synthetase. Two additional proteins, CitG and CitX, are used to convert the apo enzyme into the active holo enzyme (Schneider et al, Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not have citrate lyase activity; however, mutants deficient in molybdenum cofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD and the citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman, Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citrate lyase has been cloned, characterized and expressed in E. coli (Bekal et al., J. Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have also been identified in enterobacteria that utilize citrate as a carbon and energy source, including Salmonella typhimurium and

Klebsiella pneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins are tabulated below.

Protein ( ,cn Bank ID GI Number Organism citF AAC73716.1 1786832 Escherichia coli

citE AAC73717.2 87081764 Escherichia coli

citD AAC73718.1 1786834 Escherichia coli

citC AAC73719.2 87081765 Escherichia coli

citG AAC73714.1 1786830 Escherichia coli

citX AAC73715.1 1786831 Escherichia coli

citF CAA71633.1 2842397 Leuconostoc mesenteroides citE CAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395 Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostoc mesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citX CAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1 16763998 Salmonella typhimurium cite AAL 19573.1 16419133 Salmonella typhimurium citD NP_459064.1 16763449 Salmonella typhimurium citC NP_459616.1 16764001 Salmonella typhimurium citG NP_459611.1 16763996 Salmonella typhimurium citX NP_459612.1 16763997 Salmonella typhimurium citF CAA56217.1 565619 Klebsiella pneumoniae citE CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae Protein ( ,cn Bank ID GI Number Organism citC BAH66541.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae

[0216] Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependent

phosphorylation of acetate to acetylphosphate. Exemplary acetate kinase enzymes have been characterized in many organisms including E. coli, Clostridium acetobutylicum and

Methanosarcina thermophila (Ingram-Smith et al, J. Bacteriol. 187:2386-2394 (2005); Fox and Roseman, J. Biol. Chem. 261 : 13487-13497 (1986); Winzer et al, Microbioloy 143 (Pt 10):3279-3286 (1997)). Acetate kinase activity has also been demonstrated in the gene product of E. coli purT (Marolewski et al, Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC 2.7.2.7), for example bukl and buk2 from Clostridium acetobutylicum, also accept acetate as a substrate (Hartmanis, M.G., J. Biol. Chem. 262:617- 621 (1987)).

[0217] The formation of acetyl-CoA from acetylphosphate is catalyzed by

phosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191 :559-569 (969)). Additional acetyltransferase enzymes have been characterized in Bacillus subtilis (Rado and Hoch, Biochim. Biophys. Acta 321 : 114-125 (1973), Clostridium kluyveri (Stadtman, E., Methods Enzymol. 1 :5896-599 (1955), and Thermotoga maritima (Bock et al, J. Bacteriol. 181 : 1861-1867 (1999)). This reaction is also catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19) including the ptb gene products from

Clostridium acetobutylicum (Wiesenborn et al., App. Environ. Microbiol. 55:317-322 (1989); Walter et al, Gene 134: 107-111 (1993)). Additional ptb genes are found in butyrate- producing bacterium L2-50 (Louis et al, J. Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al, Curr. Microbiol. 42:345-349 (2001). Protein ( ,cn Bank ID GI Number Organism

Pta NP 416800.1 71152910 Escherichia coli

Pta P39646 730415 Bacillus subtilis

Pta A5N801 146346896 Clostridium kluyveri

Pta Q9X0L4 6685776 Thermotoga maritima

Ptb NP_349676 34540484 Clostridium acetobutylicum

Ptb AAR19757.1 38425288 butyrate-producing bacterium L2-50

Ptb CAC07932.1 10046659 Bacillus megaterium

[0218] The acylation of acetate to acetyl-CoA is catalyzed by enzymes with acetyl-CoA synthetase activity. Two enzymes that catalyze this reaction are AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP- forming acetyl-CoA synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J. Bacteriol. 174:6590- 6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al, Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43: 1425-1431 (2004)). ADP- forming acetyl-CoA synthetases are reversible enzymes with a generally broad substrate range (Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). Two isozymes of ADP- forming acetyl-CoA synthetases are encoded in the Archaeoglobus fulgidus genome by are encoded by AF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) also accepts acetate as a substrate and reversibility of the enzyme was demonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetate, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and . aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra (2004); Musfeldt and Schonheit, supra (2002)). Additional candidates include the succinyl- CoA synthetase encoded by sucCD in E. coli (Buck et al, Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez- Valverde et al, Appl. Environ. Microbiol. 59: 1149-1154 (1993)). The aforementioned proteins are tabulated below.

[0219] The product yields per C-mol of substrate of microbial cells synthesizing reduced fermentation products such as succinate, are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing equivalents, or electrons, can be extracted from synthesis gas components such as CO and H ₂ using carbon monoxide dehydrogenase (CODH) and hydrogenase enzymes, respectively. The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H ₂ , or water, respectively. Reduced ferredoxin and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood- Ljungdahl pathway and reductive TCA cycle enzymes.

[0220] When both feedstocks of sugar and syngas are available, the syngas components CO and H ₂ can be utilized to generate reducing equivalents by employing the hydrogenase and CO dehydrogenase. The reducing equivalents generated from syngas components will be utilized to power the glucose to succinate production pathways. Theoretically, all carbons in glucose will be conserved, thus resulting in a maximal theoretical yield to produce succinate from glucose. [0221] As described above, a combined feedstock strategy where syngas is combined with a sugar-based feedstock or other carbon substrate can greatly improve the theoretical yields. In this co-feeding appoach, syngas components H ₂ and CO, or other gaseous carbon sources, can be utilized by the hydrogenase and CO dehydrogenase to generate reducing equivalents, that can be used to power chemical production pathways in which the carbons from sugar or other carbon substrates will be maximally conserved and the theoretical yields improved. Such improvements provide environmental and economic benefits and greatly enhance sustainable chemical production.

[0222] Herein below the enzymes and the corresponding genes used for extracting redox from synags components are described. CODH is a reversible enzyme that interconverts CO and C0 ₂ at the expense or gain of electrons. The natural physiological role of the CODH in ACS/CODH complexes is to convert C0 ₂ to CO for incorporation into acetyl-CoA by acetyl- CoA synthase. Nevertheless, such CODH enzymes are suitable for the extraction of reducing equivalents from CO due to the reversible nature of such enzymes. Expressing such CODH enzymes in the absence of ACS allows them to operate in the direction opposite to their natural physiological role (that is, CO oxidation).

[0223] In thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, and several other organisms, additional CODH encoding genes are located outside of the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing equivalents) from the conversion of carbon monoxide to carbon dioxide. The M. thermoacetica gene (Genbank Accession Number: YP 430813) is expressed by itself in an operon and is believed to transfer electrons from CO to an external mediator like ferredoxin in a "Ping-pong" reaction. The reduced mediator then couples to other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H) carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals of the New York Academy of Sciences 1125: 129-136 (2008)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191 :243-247 (2000)). The resulting complex was membrane -bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al., J Bacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (Dobbek et al, Science 293: 1281-1285 (2001)). Similar ACS-free CODH enzymes can be found in a diverse array of organisms including Geobacter metallireducens GS-15, Chlorobium phaeobacteroides DSM 266, Clostridium cellulolyticum H10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 2111 , Pelobacter carbinolicus DSM 2380, C. ljungdahli and Campylobacter curvus 525.92.

Protein GenBank ID GI Number Organism

CODH (putative) YP 430813 83590804 Moorella thermoacetica

CODH-II (CooS-II) YP_358957 78044574 Carboxydothermus

hydrogenoformans

CooF YP_358958 78045112 Carboxydothermus

hydrogenoformans

CODH (putative) ZP 05390164.1 255523193 Clostridium carboxidivorans

P7

CcarbDRAFT 034 ZP 05390341.1 255523371 Clostridium carboxidivorans 1 P7

CcarbDRAFT 175 ZP 05391756.1 255524806 Clostridium carboxidivorans 6 P7

CcarbDRAFT 294 ZP 05392944.1 255526020 Clostridium carboxidivorans 4 P7

CODH YP_384856.1 78223109 Geobacter metallireducens GS- 15

Cpha266_0148 YP 910642.1 119355998 Chlorobium

(cytochrome c) phaeobacteroides DSM 266

Cpha266 0149 YP 910643.1 119355999 Chlorobium

(CODH) phaeobacteroides DSM 266

Ccel_0438 YP 002504800.1 220927891 Clostridium cellulolyticum H10

Ddes_0382 YP 002478973.1 220903661 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC (CODH)

27774

Ddes_0381 (CooC) YP_002478972.1 220903660 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774

Pcar_0057 YP 355490.1 7791767 Pelobacter carbinolicus DSM

2380

(CODH)

Pcar_0058 YP 355491.1 7791766 Pelobacter carbinolicus DSM

2380

(CooC)

Pcar_0058 YP_355492.1 7791765 Pelobacter carbinolicus DSM

2380

(HypA)

CooS (CODH) YP 001407343.1 154175407 Campylobacter curvus 525.92 CLJU_c09110 ADK13979.1 300434212 Clostridium ljungdahli

CLJU_c09100 ADK13978.1 300434211 Clostridium ljungdahli

CLJU_c09090 ADK13977.1 300434210 Clostridium ljungdahli

[0224] In some cases, hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H ₂ 0 to C0 ₂ and H ₂ (Fox et al, J Bacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al, PLoS Genet. I :e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and C0 ₂ reduction activities when linked to an electrode (Parkin et al., J Am.Chem.Soc. 129: 10328- 10329 (2007)). The protein sequences of exemplary CODH and hydrogenase genes can be identified by the following GenBank accession numbers.

Protein GenBank ID GI Number Organism

CODH-I YP 360644 7804347S Carboxydothermus (CooS-I) hydrogenoformans

CooF YP 360645 78044791 Carboxydothermus

hydrogenoformans

HypA YP 360646 78044340 Carboxydothermus

hydrogenoformans

CooH YP 360647 78043871 Carboxydothermus

hydrogenoformans

CooU YP 360648 78044023 Carboxydothermus

hydrogenoformans

CooX YP 360649 78043124 Carboxydothermus

hydrogenoformans

CooL YP 360650 78043938 Carboxydothermus

hydrogenoformans

CooK YP 360651 78044700 Carboxydothermus

hydrogenoformans

CooM YP 360652 78043942 Carboxydothermus

hydrogenoformans

CooC YP 360654.1 78043296 Carboxydothermus

hydrogenoformans

CooA-1 YP 360655.1 78044021 Carboxydothermus

hydrogenoformans

CooL AAC45118 1515468 Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum

CooU AAC45120 1515470 Rhodospirillum rubrum

CooH AAC45121 1498746 Rhodospirillum rubrum

CooF AAC45122 1498747 Rhodospirillum rubrum

CODH AAC45123 1498748 Rhodospirillum rubrum (CooS)

CooC AAC45124 1498749 Rhodospirillum rubrum

CooT AAC45125 1498750 Rhodospirillum rubrum

CooJ AAC45126 1498751 Rhodospirillum rubrum

[0225] Native to E. coli and other enteric bacteria are multiple genes encoding up to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al, J Bacteriol. 164: 1324-1331 (1985); Sawers and Boxer, Eur.JBiochem. 156:265-275 (1986); Sawers et al, J Bacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities, E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. E. coli possesses two uptake hydrogenases, Hyd-1 and Hyd-2, encoded by the hyaABCDEF and hybOABCDEFG gene clusters, respectively (Lukey et al, J. Biol. Chem. 285(6):3928-3938 (2010)). Hyd-1 is oxygen-tolerant, irreversible, and is coupled to quinone reduction via the hyaC cytochrome. Hyd-2 is sensitive to 0 ₂ , reversible, and transfers electrons to the periplasmic ferredoxin hybA which, in turn, reduces a quinone via the hybB integral membrane protein. Reduced quinones can serve as the source of electrons for fumarate reductase in the reductive branch of the TCA cycle. Reduced ferredoxins can be used by enzymes such as

NAD(P)H:ferredoxin oxidoreductases to generate NADPH or NADH. They can alternatively be used as the electron donor for reactions such as pyruvate ferredoxin oxidoreductase, AKG ferredoxin oxidoreductase, and 5, 10-methylene-H4 folate reductase.

Protein GenBank ID GI Number Organism

HybO AAC76033.1 1789371 Escherichia coli HybA AAC76032.1 1789370 Escherichia coli

HybB AAC76031.1 2367183 Escherichia coli

HybC AAC76030.1 1789368 Escherichia coli

HybD AAC76029.1 1789367 Escherichia coli

HybE AAC76028.1 1789366 Escherichia coli

HybF AAC76027.1 1789365 Escherichia coli

HybG AAC76026.1 1789364 Escherichia coli

[0226] The hydrogen- lyase systems of E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase 3 has been shown to be a reversible enzyme (Maeda et al, Appl Microbiol Biotechnol 76(5): 1035-42 (2007)). Hydrogenase activity in E. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al., Arch.Microbiol 158:444-451 (1992); Rangarajan et al, J. BacterioL 190: 1447-1458 (2008)).

Protein GenBank ID GI Number Organism

HyfA NP_416976 90111444 Escherichia coli

HyfB NP_416977 16130407 Escherichia coli

HyfC NP_416978 90111445 Escherichia coli

HyfD NP_416979 16130409 Escherichia coli

HyfE NP 416980 16130410 Escherichia coli

HyfF NP_416981 16130411 Escherichia coli

HyfG NP_416982 16130412 Escherichia coli

HyfH NP_416983 16130413 Escherichia coli

Hyfl NP_416984 16130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli

Hyf NP_416986 90111447 Escherichia coli

Protein GenBank ID GI Number Organism

HypA NP_417206 16130633 Escherichia coli

HypB NP_417207 16130634 Escherichia coli

HypC NP_417208 16130635 Escherichia coli

HypD NP_417209 16130636 Escherichia coli

HypE NP_417210 226524740 Escherichia coli

HypF NP_417192 16130619 Escherichia coli

[0227] The M. thermoacetica hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M. thermoacetica can grow with C0 ₂ as the exclusive carbon source indicating that reducing equivalents are extracted from H ₂ to enable acetyl- CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J. Bacteriol. 150:702-709 (1982); Drake and Daniel, Res. Microbiol. 155:869-883 (2004); Kellum and Drake, J. Bacteriol. 160:466-469 (1984)) (see Figure 4). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. The protein sequences encoded for by these genes are identified by the following GenBank accession numbers.

[0228] Proteins in M. thermoacetica whose genes are homologous to the E. coli hyp genes are shown below.

[0229] Proteins in M. thermoacetica that are homologous to the E. coli Hydrogenase 3 and/or 4 proteins are listed in the following table.

Protein GenBank ID GI Number Organism

Moth_2182 YP 431014 83591005 Moorella thermoacetica

Moth_2183 YP 431015 83591006 Moorella thermoacetica Moth_2184 YP 431016 83591007 Moorella thermoacetica

Moth_2185 YP 431017 83591008 Moorella thermoacetica

Moth_2186 YP 431018 83591009 Moorella thermoacetica

Moth_2187 YP 431019 83591010 Moorella thermoacetica

Moth_2188 YP 431020 83591011 Moorella thermoacetica

Moth_2189 YP 431021 83591012 Moorella thermoacetica

Moth_2190 YP_431022 83591013 Moorella thermoacetica

Moth_2191 YP 431023 83591014 Moorella thermoacetica

Moth_2192 YP 431024 83591015 Moorella thermoacetica

[0230] In addition, several gene clusters encoding hydrogenase functionality are present in M. thermoacetica and their corresponding protein sequences are provided below.

Protein GenBank ID GI Number Organism

Moth_0439 YP_429313 83589304 Moorella thermoacetica

Moth_0440 YP_429314 83589305 Moorella thermoacetica

Moth_0441 YP_429315 83589306 Moorella thermoacetica

Moth_0442 YP 429316 83589307 Moorella thermoacetica

Moth_0809 YP 429670 83589661 Moorella thermoacetica

Moth_0810 YP 429671 83589662 Moorella thermoacetica

Moth_0811 YP_429672 83589663 Moorella thermoacetica

Moth_0812 YP_429673 83589664 Moorella thermoacetica

Moth_0814 YP_429674 83589665 Moorella thermoacetica

Moth_0815 YP_429675 83589666 Moorella thermoacetica

Moth_0816 YP 429676 83589667 Moorella thermoacetica

Moth_1193 YP 430050 83590041 Moorella thermoacetica

Moth_1194 YP 430051 83590042 Moorella thermoacetica

Moth_1195 YP 430052 83590043 Moorella thermoacetica

Moth_1196 YP 430053 83590044 Moorella thermoacetica

Moth_1717 YP 430562 83590553 Moorella thermoacetica

Moth_1718 YP 430563 83590554 Moorella thermoacetica

Moth_1719 YP 430564 83590555 Moorella thermoacetica

Moth_1883 YP_430726 83590717 Moorella thermoacetica

Moth_1884 YP_430727 83590718 Moorella thermoacetica

Moth_1885 YP_430728 83590719 Moorella thermoacetica

Moth_1886 YP 430729 83590720 Moorella thermoacetica

Moth_1887 YP 430730 83590721 Moorella thermoacetica

Moth_1888 YP 430731 83590722 Moorella thermoacetica

Moth_1452 YP 430305 83590296 Moorella thermoacetica

Moth_1453 YP 430306 83590297 Moorella thermoacetica Moth_1454 YP 430307 83590298 Moorella thermoacetica

[0231] Ralstonia eutropha HI 6 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an "02- tolerant" hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type

cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al, Eur. J. Biochem. 248, 179-186 (1997)). R. eutropha also contains an 0 ₂ -tolerant soluble hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).

HoxE NP_953767.1 39997816 Geobacter sulfurreducens

HoxF NP_953766.1 39997815 Geobacter sulfurreducens

HoxU NP_953765.1 39997814 Geobacter sulfurreducens

HoxY NP_953764.1 39997813 Geobacter sulfurreducens

HoxH NP_953763.1 39997812 Geobacter sulfurreducens

GSU2717 NP_953762.1 39997811 Geobacter sulfurreducens

HoxE NP_441418.1 16330690 Synechocystis str. PCC 6803

HoxF NP_441417.1 16330689 Synechocystis str. PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803 function

HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803

HoxY NP_441414.1 16330686 Synechocystis str. PCC 6803

Unknown NP_441413.1 16330685 Synechocystis str. PCC 6803 function

Unknown NP_441412.1 16330684 Synechocystis str. PCC 6803 function

HoxH NP_441411.1 16330683 Synechocystis str. PCC 6803

HypF NP_484737.1 17228189 Nostoc sp. PCC 7120

HypC NP_484738.1 17228190 Nostoc sp. PCC 7120

HypD NP_484739.1 17228191 Nostoc sp. PCC 7120

Unknown NP_484740.1 17228192 Nostoc sp. PCC 7120 function

HypE NP_484741.1 17228193 Nostoc sp. PCC 7120

HypA NP_484742.1 17228194 Nostoc sp. PCC 7120

HypB NP_484743.1 17228195 Nostoc sp. PCC 7120

32] Genes encoding hydrogenase enzymes from C ljungdahli are shown below.

Protein GenBank ID GI Number Organism

CLJU_c20290 ADK15091.1 300435324 Clostridium ljungdahli

CLJU_c07030 ADK13773.1 300434006 Clostridium ljungdahli

CLJU_c07040 ADK13774.1 300434007 Clostridium ljungdahli

CLJU_c07050 ADK13775.1 300434008 Clostridium ljungdahli

CLJU_c07060 ADK13776.1 300434009 Clostridium ljungdahli

CLJU_c07070 ADK13777.1 300434010 Clostridium ljungdahli

CLJU_c07080 ADK13778.1 300434011 Clostridium ljungdahli

CLJU_cl4730 ADK14541.1 300434774 Clostridium ljungdahli

CLJU_cl4720 ADK14540.1 300434773 Clostridium ljungdahli

CLJU_cl4710 ADK14539.1 300434772 Clostridium ljungdahli

CLJU_cl4700 ADK14538.1 300434771 Clostridium ljungdahli CLJU_c28670 ADK15915.1 300436148 Clostridium ljungdahli

CLJU_c28660 ADK15914.1 300436147 Clostridium ljungdahli

CLJU_c28650 ADK15913.1 300436146 Clostridium ljungdahli

CLJU_c28640 ADK15912.1 300436145 Clostridium ljungdahli

[0233] Several enzymes and the corresponding genes used for fixing carbon dioxide to either pyruvate or phosphoenolpyruvate to form the TCA cycle intermediates, oxaloacetate or malate are described below.

[0234] Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed by

phosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes are encoded by ppc in E. coli (Kai et al, Arch. Biochem. Biophys. 414: 170-179 (2003), ppcA in

Methylobacterium extorquens AMI (Arps et al, J. Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacterium glutamicum (Eikmanns et al, Mol. Gen. Genet. 218:330-339 (1989).

[0235] An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase, which simultaneously forms an ATP while carboxylating PEP. In most organisms PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP. S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al, FEBS Lett. 258:313- 316 (1989). E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher K _m for bicarbonate of PEP carboxykinase (Kim et al., Appl. Environ. Microbiol. 70: 1238-1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al, J. Microbiol. Biotechnol. 16: 1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHC0 ₃ concentrations. Mutant strains of E. coli can adopt Pck as the dominant C0 ₂ -fixing enzyme following adaptive evolution (Zhang et al, supra, 2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobio spirillum succiniciproducens (Laivenieks et al., Appl. Environ. Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim et al. supra). The PEP carboxykinase enzyme encoded by Haemophilus influenza is effective at forming oxaloacetate from PEP.

[0236] Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate to oxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes are encoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun. 176: 1210-1217 (1991) and PYC2 (Walker et al, supra) in Saccharomyces cerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay and Purwantini, Biochim. Biophys. Acta 1475: 191-206 (2000)).

[0237] Malic enzyme can be applied to convert C0 ₂ and pyruvate to malate at the expense of one reducing equivalent. Malic enzymes for this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic enzyme (NADP-dependent). For example, one of the E. coli malic enzymes (Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higher activity can be expressed to enable the conversion of pyruvate and C0 ₂ to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzyme allows the high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP is generated in the formation of pyruvate or by the phosphotransferase system for glucose transport. Although malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded by maeA, has been demonstrated to increase succinate production in E. coli while restoring the lethal Apfl-AldhA phenotype under anaerobic conditions by operating in the carbon- fixing direction (Stols and Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was made upon overexpressing the malic enzyme from Ascaris suum in E. coli (Stols et al, Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, is NADP-dependent and also decarboxylates oxaloacetate and other alpha-keto acids (Iwakura et al, J. Biochem. 85(5): 1355-65 (1979)).

[0238] The enzymes used for converting oxaloacetate (formed from, for example, PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase) or malate (formed from, for example, malic enzyme or malate dehydrogenase) to succinyl-CoA via the reductive branch of the TCA cycle are malate dehydrogenase, fumarate dehydratase (fumarase), fumarate reductase, and succinyl-CoA transferase. The genes for each of the enzymes are described herein above.

[0239] Enzymes, genes and methods for engineering pathways from succinyl-CoA to various products into a microorganism are now known in the art. The additional reducing equivalents obtained from CO and/or H ₂ , as disclosed herein, improve the yields of succinate when utilizing carbohydrate-based feedstock.

[0240] Enzymes, genes and methods for engineering pathways from glycolysis intermediates to various products into a microorganism are known in the art. The additional reducing equivalents obtained from CO and H ₂ , as described herein, improve the yields of all these products on carbohydrates.

EXAMPLE V

Methods for Handling CO and Anaerobic Cultures

[0241] This example describes methods used in handling CO and anaerobic cultures.

[0242] A. Handling of CO in small quantities for assays and small cultures. CO is an odorless, colorless and tasteless gas that is a poison. Therefore, cultures and assays that utilized CO required special handling. Several assays, including CO oxidation, acetyl-CoA synthesis, CO concentration using myoglobin, and CO tolerance/utilization in small batch cultures, called for small quantities of the CO gas that were dispensed and handled within a fume hood. Biochemical assays called for saturating very small quantities (<2 mL) of the biochemical assay medium or buffer with CO and then performing the assay. All of the CO handling steps were performed in a fume hood with the sash set at the proper height and blower turned on; CO was dispensed from a compressed gas cylinder and the regulator connected to a Schlenk line. The latter ensures that equal concentrations of CO were dispensed to each of several possible cuvettes or vials. The Schlenk line was set up containing an oxygen scrubber on the input side and an oil pressure release bubbler and vent on the other side. Assay cuvettes were both anaerobic and CO-containing. Threfore, the assay cuvettes were tightly sealed with a rubber stopper and reagents were added or removed using gas-tight needles and syringes. Secondly, small (-50 mL) cultures were grown with saturating CO in tightly stoppered serum bottles. As with the biochemical assays, the CO- saturated microbial cultures were equilibrated in the fume hood using the Schlenk line setup. Both the biochemical assays and microbial cultures were in portable, sealed containers and in small volumes making for safe handling outside of the fume hood. The compressed CO tank was adjacent to the fume hood.

[0243] Typically, a Schlenk line was used to dispense CO to cuvettes, each vented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gage disposable syringe needles and were vented with the same. An oil bubbler was used with a CO tank and oxygen scrubber. The glass or quartz spectrophotometer cuvettes have a circular hole on top into which a Kontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unit was positioned proximal to the fume hood.

[0244] B. Handling of CO in larger quantities fed to large-scale cultures. Fermentation cultures are fed either CO or a mixture of CO and H ₂ to simulate syngas as a feedstock in fermentative production. Therefore, quantities of cells ranging from 1 liter to several liters can include the addition of CO gas to increase the dissolved concentration of CO in the medium. In these circumstances, fairly large and continuously administered quantities of CO gas are added to the cultures. At different points, the cultures are harvested or samples removed. Alternatively, cells are harvested with an integrated continuous flow centrifuge that is part of the fermenter. [0245] The fermentative processes are carried out under anaerobic conditions. In some cases, it is uneconomical to pump oxygen or air into fermenters to ensure adequate oxygen saturation to provide a respiratory environment. In addition, the reducing power generated during anaerobic fermentation may be needed in product formation rather than respiration. Furthermore, many of the enzymes for various pathways are oxygen-sensitive to varying degrees. Classic acetogens such as M. thermoacetica are obligate anaerobes and the enzymes in the Wood-Ljungdahl pathway are highly sensitive to irreversible inactivation by molecular oxygen. While there are oxygen-tolerant acetogens, the repertoire of enzymes in the Wood- Ljungdahl pathway might be incompatible in the presence of oxygen because most are metallo-enzymes, key components are ferredoxins, and regulation can divert metabolism away from the Wood-Ljungdahl pathway to maximize energy acquisition. At the same time, cells in culture act as oxygen scavengers that moderate the need for extreme measures in the presence of large cell growth.

[0246] C. Anaerobic chamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA). Conditions included an 0 ₂ concentration of 1 ppm or less and 1 atm pure N ₂ . In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 0 ₂ electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume >5mL were sparged with pure N ₂ prior to

introduction into the chamber. Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to changes in oxygen levels. The chamber's pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve.

[0247] The anaerobic chambers achieved levels of 0 ₂ that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions. In an alternative anaerobic chamber configuration, platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler. Instead of using instrument-based 0 ₂ monitoring, test strips can be used instead. [0248] D. Anaerobic microbiology. Small cultures were handled as described above for CO handling. In particular, serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with nitrogen for ~30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave. The sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle. Prior to addition of reducing agents, the bottles are equilibrated for 30 - 60 minutes with nitrogen (or CO depending upon use). A reducing agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine-HCl is added. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B 12 (10 μΜ cyanocobalamin), nickel chloride (NiCl ₂ , 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and ferrous ammonium sulfate (final concentration needed is 100 μΜ— made as 100-lOOOx stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pAl-lacOl promoter in the vectors was performed by addition of isopropyl β-D-l- thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs.

[0249] Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once all medium components and cells are added, the bottles are incubated in an incubator in room air but with continuous nitrogen sparging into the bottles.

EXAMPLE VI CO oxidation (CODH) Assay

[0250] This example describes assay methods for measuring CO oxidation (CO dehydrogenase; CODH).

[0251] The 7 gene CODH/ ACS operon of Moorella thermoacetica was cloned into E. coli expression vectors. The intact ~10 kbp DNA fragment was cloned, and it is likely that some of the genes in this region are expressed from their own endogenous promoters and all contain endogenous ribosomal binding sites. These clones were assayed for CO oxidation, using an assay that quantitatively measures CODH activity. Antisera to the M. thermoacetica gene products was used for Western blots to estimate specific activity. M. thermoacetica is Gram positive, and ribosome binding site elements are expected to work well in E. coli. This activity, described below in more detail, was estimated to be -1/50th of the M. thermoacetica specific activity. It is possible that CODH activity of recombinant E. coli cells could be limited by the fact that thermoacetica enzymes have temperature optima around 55°C. Therefore, a mesophilic CODH/ ACS pathway could be advantageous such as the close relative of Moorella that is mesophilic and does have an apparently intact CODH/ ACS operon and a Wood-Ljungdahl pathway, Desulfitobacterium hafniense. Acetogens as potential host organisms include, but are not limited to, Rhodospirillum rubrum, Moorella thermoacetica and Desulfitobacterium hafniense.

[0252] CO oxidation is both the most sensitive and most robust of the CODH/ ACS assays. It is likely that an E. co/z ^' -based syngas using system will ultimately need to be about as anaerobic as Clostridial (i.e., Moorella) systems, especially for maximal activity.

Improvement in CODH should be possible but will ultimately be limited by the solubility of CO gas in water.

[0253] Initially, each of the genes was cloned individually into expression vectors.

Combined expression units for multiple subunits/1 complex were generated. Expression in E. coli at the protein level was determined. Both combined M. thermoacetica CODH/ ACS operons and individual expression clones were made. [0254] CO oxidation assay. This assay is one of the simpler, reliable, and more versatile assays of enzymatic activities within the Wood-Ljungdahl pathway and tests CODH

(Seravalli et al, Biochemistry 43:3944-3955 (2004)). A typical activity ofM thermoacetica CODH specific activity is 500 U at 55°C or ~60U at 25°C. This assay employs reduction of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered, anaerobic, glass cuvettes.

[0255] In more detail, glass rubber stoppered cuvettes were prepared after first washing the cuvette four times in deionized water and one time with acetone. A small amount of vacuum grease was smeared on the top of the rubber gasket. The cuvette was gassed with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle. A volume of 0.98 mlL of reaction buffer (50 mM Hepes, pH 8.5, 2mM dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust needled, and 100%CO. Methyl viologen (CH ₃ viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final concentration. When methyl viologen was added, an 18 Ga needle (partial) was used as a jacket to facilitate use of a Hamilton syringe to withdraw the CH ₃ viologen. 4 -5 aliquots were drawn up and discarded to wash and gas equilibrate the syringe. A small amount of sodium dithionite (0.1 M stock) was added when making up the CH ₃ viologen stock to slightly reduce the CH ₃ viologen. The temperature was equilibrated to 55°C in a heated Olis spectrophotometer (Bogart GA). A blank reaction (CH ₃ viologen + buffer) was run first to measure the base rate of CH ₃ viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91 (CODH- ACS operon of M. thermoacetica with and without, respectively, the first cooC). 10 microliters of extract were added at a time, mixed and assayed. Reduced CH ₃ viologen turns purple. The results of an assay are shown in Table I.

Table I. Crude extract CO Oxidation Activities.

[0256] Mta98/Mta99 are E. coli MG1655 strains that express methanol methyltransferase genes from thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M. thermoacetica CODH operons.

[0257] If ~ 1% of the cellular protein is CODH, then these figures would be

approximately 100X less than the 500 U/mg activity of pure M. thermoacetica CODH.

Actual estimates based on Western blots are 0.5% of the cellular protein, so the activity is about 50X less than for thermoacetica CODH. Nevertheless, this experiment

demonstrates CO oxidation activity in recombinant E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation (CH ₃ viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce CH ₃ viologen.

[0258] To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGE followed by Western blot analyses were performed on the same cell extracts used in the CO oxidation, ACS, methyltransferase, and corrinoid Fe-S assays. The antisera used were polyclonal to purified M. thermoacetica CODH- ACS and Mtr proteins and were visualized using an alkaline phosphatase-linked goat-anti-rabbit secondary antibody. The Westerns were performed and results are shown in Figure 6. The amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison to the control lanes. Expression of CODH- ACS operon genes including 2 CODH subunits and the methyltransferase were confirmed via Western blot analysis. Therefore, the recombinant E. coli cells express multiple components of a 7 gene operon. In addition, both the methyltransferase and corrinoid iron sulfur protein were active in the same recombinant E. coli cells. These proteins are part of the same operon cloned into the same cells.

[0259] The CO oxidation assays were repeated using extracts of Moorella thermoacetica cells for the positive controls. Though CODH activity in E. coli ACS90 and ACS91 was measurable, it was at about 130 - 150 X lower than the M. thermoacetica control. The results of the assay are shown in Figure 7. Briefly, cells (M thermoacetica or E. coli with the CODH/ ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared as described above. Assays were performed as described above at 55°C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course.

[0260] These results describe the CO oxidation (CODH) assay and results. Recombinant E. coli cells expressed CO oxidation activity as measured by the methyl viologen reduction assay.

EXAMPLE VII

E. coli CO Tolerance Experiment and CO Concentration Assay (myoglobin assay)

[0261] This example describes the tolerance of E. coli for high concentrations of CO.

[0262] To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO, cultures were set up in 120 ml serum bottles with 50 ml of Terrific Broth medium (plus reducing solution, NiCl ₂ , Fe(II)NH ₄ S0 ₄ , cyanocobalamin, IPTG, and chloramphenicol) as described above for anaerobic microbiology in small volumes. One half of these bottles were equilibrated with nitrogen gas for 30 min. and one half was equilibrated with CO gas for 30 min. An empty vector (pZA33) was used as a control, and cultures containing the pZA33 empty vector as well as both ACS90 and ACS91 were tested with both N ₂ and CO. All were inoculated and grown for 36 hrs with shaking (250 rpm) at 37°C. At the end of the 36 hour period, examination of the flasks showed high amounts of growth in all. The bulk of the observed growth occurred overnight with a long lag. [0263] Given that all cultures appeared to grow well in the presence of CO, the final CO concentrations were confirmed. This was performed using an assay of the spectral shift of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low wavelength and need to record a whole spectrum from 300 nm on upwards, quartz cuvettes must be used. CO concentration is measured against a standard curve and depends upon the Henry's Law constant for CO of maximum water solubility = 970 micromolar at 20°C and 1 atm.

[0264] For the myoglobin test of CO concentration, cuvettes were washed 10X with water, IX with acetone, and then stoppered as with the CODH assay. N ₂ was blown into the cuvettes for ~10 min. A volume of 1 ml of anaerobic buffer (HEPES, pH 8.0, 2mM DTT) was added to the blank (not equilibrated with CO) with a Hamilton syringe. A volume of 10 microliter myoglobin (~1 mM— can be varied, just need a fairly large amount) and 1 microliter dithionite (20 mM stock) were added. A CO standard curve was made using CO saturated buffer added at 1 microliter increments. Peak height and shift was recorded for each increment. The cultures tested were pZA33/CO, ACS90/CO, and ACS91/CO. Each of these was added in 1 microliter increments to the same cuvette. Midway through the experiment a second cuvette was set up and used. The results are shown in Table II.

Table II. Carbon Monoxide Concentrations, 36 hrs.

Strain and Growth Conditions Final CO concentration (micromolar) :

PZA33-CO 930

ACS90-CO 638

494

734

883

ave 687

SD 164

ACS91-CO 728

812

760

611

ave. 728

SD 85 [0265] The results shown in Table II indicate that the cultures grew whether or not a strain was cultured in the presence of CO or not. These results indicate that E. coli can tolerate exposure to CO under anaerobic conditions and that E. coli cells expressing the CODH-ACS operon can metabolize some of the CO.

[0266] These results demonstrate that E. coli cells, whether expressing CODH/ACS or not, were able to grow in the presence of saturating amounts of CO. Furthermore, these grew equally well as the controls in nitrogen in place of CO. This experiment demonstrated that laboratory strains of E. coli are insensitive to CO at the levels achievable in a syngas project performed at normal atmospheric pressure. In addition, preliminary experiments indicated that the recombinant E. coli cells expressing CODH/ACS actually consumed some CO, probably by oxidation to carbon dioxide.

EXAMPLE VIII

Exemplary Carboxylic Acid Reductases

[0267] This example describes the use of carboxylic acid reductases to carry out the conversion of a caroboxylic acid to an aldehyde.

[0268] 1.2.1.e Acid reductase. The conversion of unactivated acids to aldehydes can be carried out by an acid reductase. Examples of such conversions include, but are not limited, the conversion of 4-hydroxybutyrate, succinate, alpha-ketoglutarate, and 4-aminobutyrate to 4-hydroxybutanal, succinate semialdehyde, 2,5-dioxopentanoate, and 4-aminobutanal, respectively. One notable carboxylic acid reductase can be found in Nocardia iowensis which catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). This enzyme is encoded by the car gene and was cloned and functionally expressed in E. coli (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)). Expression of the npt gene product improved activity of the enzyme via post-transcriptional modification. The npt gene encodes a specific phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme. The natural substrate of this enzyme is vanillic acid, and the enzyme exhibits broad acceptance of aromatic and aliphatic substrates (Venkitasubramanian et al., in Biocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, FL.

(2006)). Gene Accession No. GI No. Organism

Nocardia iowensis (sp.

car AAR91681.1 40796035 NRRL 5646)

Nocardia iowensis (sp.

npt ABI83656.1 114848891 NRRL 5646)

[0269] Additional car and npt genes can be identified based on sequence homology.

[0270] An additional enzyme candidate found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme is believed to convert 3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3-amino-4- hydroxybenzoic acid metabolism (Suzuki, et al, J. Antibiot. 60(6):380-387 (2007)). Co- expression of griC and griD with SGR 665, an enzyme similar in sequence to the Nocardia iowensis npt, can be beneficial.

Gene Accession No. GI No. Organism

Streptomyces griseus subsp. griC 182438036 YP 001825755.1

griseus NBRC 13350

Streptomyces griseus subsp. griD 182438037 YP 001825756.1

griseus NBRC 13350

Mycobacterium smegmatis

MSMEG 956 YP_887275.1 YP_887275.1

MC2 155

Mycobacterium smegmatis

MSMEG 739 YP_889972.1 118469671

MC2 155

Mycobacterium smegmatis

MSMEG 648 YP_886985.1 118471293

MC2 155

Mycobacterium avium subsp.

MAP 1040c NP_959974.1 41407138 paratuberculosis K-10 Mycobacterium avium subsp.

MAP2899c NP_961833.1 41408997

paratuberculosis K-10

YP 001850422.

MMAR 117 183982131 Mycobacterium marinum M

1

YP 001851230.

MMAR 936 183982939 Mycobacterium marinum M

1

YP 001850220.

MMARJ916 183981929 Mycobacterium marinum M

1

TpauDRAFT 3 Tsukamurella paurometabola

ZP 04027864.1 227980601

3060 DSM 20162

TpauDRAFT 2 Tsukamurella paurometabola

ZP 04026660.1 227979396

0920 DSM 20162

CPCC7001 13

ZP 05045132.1 254431429 Cyanobium PCC7001

20

DDBDRAFT 01

XP 636931.1 66806417 Dictyostelium discoideum AX4 87729

[0271] An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and requires activation by a PPTase. Enzyme candidates for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al, Gene 98: 141- 145 (1991)), Candida albicans (Guo et al, Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al, Curr. Genet. 28: 131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al, Yeast 21 : 1279- 1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al, J. Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P.

chrysogenum PPTase has not been identified to date.

Gene Accession No. GI No. Organism

LYS2 AAA34747.1 171867 Saccharomyces cerevisiae

LYS5 P501 13.1 1708896 Saccharomyces cerevisiae

LYS2 AAC02241.1 2853226 Candida albicans

LYS5 AAO26020.1 28136195 Candida albicans

Lyslp P40976.3 13124791 Schizosaccharomyces pombe

Lys7p Q 10474.1 1723561 Schizosaccharomyces pombe

Lys2 CAA74300.1 3282044 Penicillium chrysogenum [0272] Cloning and Expression of Carboxylic Acid Reductase. Escherichia coli is used as a target organism to engineer the pathway for succinate. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing succinate. E. coli is amenable to genetic manipulation and is known to be capable of producing various intermediates and products effectively under various oxygenation conditions.

[0273] To generate a microbial organism strain such as an E. coli strain engineered to produce succinate, nucleic acids encoding a carboxylic acid reductase and

phosphopantetheine transferase are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular, car genes from Nocardia iowensis (designated 720), Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium avium subspecies paratuberculosis K-10 (designated 891) and Mycobacterium marinum M (designated 892) were cloned into pZS* 13 vectors

(Expressys, Ruelzheim, Germany) under control of PAl/lacO promoters. The npt

(ABI83656.1) gene (i.e., 721) was cloned into the pKJL33S vector, a derivative of the original mini-F plasmid vector PML31 under control of promoters and ribosomal binding sites similar to those used in pZS* 13.

[0274] The car gene (GNM_720) was cloned by PCR from Nocardia genomic DNA. Its nucleic acid and protein sequences are shown in Figures 8 A and 8B, respectively. A codon- optimized version of the npt gene (GNM_721) was synthesized by GeneArt (Regensburg, Germany). Its nucleic acid and protein sequences are shown in Figures 9A and 9B, respectively. The nucleic acid and protein sequences for the Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium avium subspecies paratuberculosis K-10 (designated 891) and Mycobacterium marinum M (designated 892) genes and enzymes can be found in Figures 10, 11, and 12, respectively. The plasmids are transformed into a host cell to express the proteins and enzymes required for succinate production.

[0275] Additional CAR variants were generated. A codon optimized version of CAR 891 was generated and designated 891GA. The nucleic acid and amino acid sequences of CAR 891GA are shown in Figures 13A and 13B, respectively. Over 2000 CAR variants were generated. In particular, all 20 amino acid combinations were made at positions V295, M296, G297, G391, G421, D413, G414, Y415, G416, and S417, and additional variants were tested as well. Exemplary CAR variants include: E16K; Q95L; L100M; A101 IT; K823E; T941S; H15Q; D198E; G446C; S392N; F699L; V883I; F467S; T987S; R12H; V295G;

V295A; V295S; V295T; V295C; V295V; V295L; V295I; V295M; V295P; V295F; V295Y; V295W; V295D; V295E; V295N; V295Q; V295H; V295K; V295R; M296G; M296A;

M296S; M296T; M296C; M296V; M296L; M296I; M296M; M296P; M296F; M296Y;

M296W; M296D; M296E; M296N; M296Q; M296H; M296K; M296R; G297G; G297A; G297S; G297T; G297C; G297V; G297L; G297I; G297M; G297P; G297F; G297Y; G297W; G297D; G297E; G297N; G297Q; G297H; G297K; G297R; G391G; G391A; G391S; G391T; G391C; G391V; G391L; G391I; G391M; G391P; G391F; G391Y; G391W; G391D; G391E; G391N; G391Q; G391H; G391K; G391R; G421G; G421A; G421S; G421T; G421C; G421V; G421L; G421I G421M; G421P; G421F; G421Y; G421W; G421D; G421E; G421N; G421Q; G421H; G421K; G421R; D413G; D413A; D413S; D413T; D413C; D413V; D413L; D413I; D413M; D413P; D413F; D413Y; D413W; D413D; D413E; D413N; D413Q; D413H;

D413K; D413R; G414G; G414A; G414S; G414T; G414C; G414V; G414L; G414I; G414M; G414P; G414F; G414Y; G414W; G414D; G414E; G414N; G414Q; G414H; G414K;

G414R; Y415G; Y415A; Y415S; Y415T; Y415C; Y415V; Y415L; Y415I; Y415M; Y415P; Y415F; Y415Y; Y415W; Y415D; Y415E; Y415N; Y415Q; Y415H; Y415K; Y415R;

G416G; G416A; G416S; G416T; G416C; G416V; G416L; G416I; G416M; G416P; G416F; G416Y; G416W; G416D; G416E; G416N; G416Q; G416H; G416K; G416R; S417G;

S417A; S417S; S417T; S417C; S417V S417L; S417I; S417M; S417P; S417F; S417Y;

S417W; S417D; S417E; S417N; S417Q; S417H; S417K; and S417R.

[0276] The CAR variants were screened for activity, and numerous CAR variants were found to exhibit CAR activity.

[0277] This example describes the use of CAR for converting carboxylic acids to aldehydes.

[0278] Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.