MICROORGANISMS FOR PRODUCING ISOBUTANOL AND METHODS RELATED

THERETO

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S. Serial Nos. 61/500,124, filed June 22, 201 1, and 61/502,708, filed June 29, 201 1, the contents of which are herein

incorporated by reference in their entirety.

BACKGROUND

[0001 ] The present invention relates generally to biosynthetic processes and organisms capable of producing organic compounds. More specifically, the invention relates to non- naturally occurring organisms that can produce the commodity chemicals isopropanol, w-butanol, or isobutanol.

[0002] Isopropanol is a colorless, flammable, three-carbon alcohol that mixes completely with most solvents, including water. The largest use for isopropanol is as a solvent, including its well known yet small use as "rubbing alcohol," which is a mixture of isopropanol and water. As a solvent, isopropanol is found in many everyday products such as paints, lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, and pharmaceuticals. Low-grade isopropanol is also used in motor oils. The second largest use is as a chemical intermediate for the production of isopropylamines (e.g. in agricultural products), isopropylethers, and isopropyl esters. Isopropanol is manufactured by two petrochemical routes. The predominant process entails the hydration of propylene either with or without sulfuric acid catalysis. Secondarily, isopropanol is produced via hydrogenation of acetone, which is a byproduct formed in the production of phenol and propylene oxide. High-priced propylene is currently driving costs up and margins down throughout the chemical industry motivating the need for an expanded range of low cost feedstocks.

[0003] Butanol, or equivalently, w-butanol, is a four carbon alcohol that is currently manufactured almost exclusively through the use of petrochemical raw materials. The main petrochemical process entails carbonylation of propylene to butyraldehyde, followed by catalytic hydrogenation to butanol. The demand for butanol is driven by its use for production of butyl acrylate and butyl methacrylate, both of which are employed in emulsified and solution polymers used in water-based latex coatings, enamels, and lacquers. Other application include its use as an intermediate for large volume chemicals such as butyl acetate and glycol butyl ethers, as well as it direct use as a solvent. Butanol also is being considered for potential application as a biofuel derived from renewable resources. Butanol has a wide range of properties that make it better suited as a fuel than ethanol. For example, butanol has higher energy content, lower volatility and hygroscopicity, can be shipped through pipeline infrastructure, can be used directly without blending, and can be blended with diesel or biodiesel.

[0004] Isobutanol is another colorless, flammable, four carbon alcohol that is being aggressively pursued as a biofuel. Currently, its major application is as a starting material for isobutyl acetate, a common solvent used in the production of lacquer and coatings and also as a flavoring agent in the food industry. Isobutyl esters are used in plastics, rubbers, and other dispersions. Additional applications for isobutanol include its use as a solvent in paint, varnish removers, and inks. Methods for isobutanol synthesis from petroleum derived feedstocks include oxo synthesis (Weber et al., Industrial & Engineering Chemistry Research, 62:33-37 (1970)) and Guerbet condensation of methanol with «-propanol (Carlini et al., J. of Molecular Catalysis A: Chemical, 220:215-220 (2004);Carlini et al., J. of Molecular Catalysis A: Chemical, 184:273- 280 (2002);Carlini et al., J. of Molecular Catalysis A: Chemical, 200: 137-146 (2003);Carlini et al., J. of Molecular Catalysis A: Chemical, 206:409-418 (2003)).

[0005] Thus, there exists a need to develop microorganisms and methods of their use to produce isopropanol, w-butanol, or isobutanol using low cost feedstocks. The present invention satisfies this need and provides related advantages as well.

SUMMARY

[0006] In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having an isopropanol pathway having at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol. The isopropanol pathway includes an enzyme selected from the group consisting of a 4-hydroxybutyryl-CoA dehydratase, a crotonase, a 3- hydroxybutyryl-CoA dehydrogenase, an acetoacetyl-CoA synthetase, an acetyl- CoA:acetoacetate-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase, and an acetone reductase.

[0007] In other embodiments, the present invention provides a method for producing isopropanol that includes culturing such a non-naturally occurring microbial organism having an isopropanol pathway under conditions and for a sufficient period of time to produce isopropanol.

[0008] In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a «-butanol pathway having at least one exogenous nucleic acid encoding a «-butanol pathway enzyme expressed in a sufficient amount to produce «-butanol. The «-butanol pathway comprising an enzyme selected from the group consisting of a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, a butyryl- CoA reductase (aldehyde forming), a butyraldehyde reductase, and a butyryl-CoA reductase (alcohol forming).

[0009] In some embodiments, the present invention provides a method for producing n- butanol comprising culturing a non-naturally occurring microbial organism having an «-butanol pathway, under conditions and for a sufficient period of time to produce «-butanol.

[0010] In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having an isobutanol pathway having at least one exogenous nucleic acid encoding an isobutanol pathway enzyme expressed in a sufficient amount to produce isobutanol. The isobutanol pathway includes an enzyme selected from the group consisting of a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl-CoA mutase, a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, an isobutyryl-CoA reductase (aldehyde forming), an isobutyraldehyde reductase, and an isobutyryl-CoA reductase (alcohol forming).

[0011] In some embodiments, the present invention provides a method for producing isobutanol that includes culturing a non-naturally occurring microbial organism having an isobutanol pathway, under conditions and for a sufficient period of time to produce isobutanol.

[0012] In some embodiments, the present invention provides a non-naturally occurring microbial organism, that includes a microbial organism having an isobutanol pathway that includes at least one exogenous nucleic acid encoding a isobutanol pathway enzyme expressed in a sufficient amount to produce isobutanol; thenon-naturally occurring microbial organism further includes:

[0013] (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha- ketoglutarate: ferredoxin oxidoreductase;

[0014] (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate: ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an ¾ hydrogenase; or

[0015] (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an ¾ hydrogenase, and combinations thereof;

[0016] wherein when the non-naturally occurring microbial organism includes an isobutanol pathway that converts 4-hydroxybutryl-CoA to isobutanol, a 4-hydroxybutyryl pathway is selected from:

[0017] (i) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (aldehyde forming), a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[0018] (ii) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (alcohol forming), a 4-Hydroxybutyrate kinase, a

Phosphotrans-4-hydroxybutyrylase;

[0019] (iii) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (aldehyde forming), a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; [0020] (iv) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (alcohol forming), a 4-Hydroxybutyryl-CoA transferase, or 4- Hydroxybutyryl-CoA synthetase;

[0021] (v) an Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase), a 4-Hydroxybutyrate dehydrogenase, a 4-Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[0022] (vi) an Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase), a 4-Hydroxybutyrate dehydrogenase, a 4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[0023] (vii) a Succinate reductase, a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[0024] (viii) a Succinate reductase, a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[0025] wherein the isobutanol pathway includes a pathway selected from:

[0026] (a) a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl- CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase;

[0027] (b) a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl- CoA mutase, and an isobutyryl-CoA reductase (alcohol forming);

[0028] (c) a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase;

[0029] (d) a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, and an isobutyryl-CoA reductase (alcohol forming); [0030] (e) an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA reductase (butyryl-CoA forming) an isobutyryl-CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and a branched-chain alcohol dehydrogenase;

[0031] (f) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase;

[0032] (g) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a valine dehydrogenase or transaminase, a valine decarboxylase, an omega transaminase, and a branched-chain alcohol dehydrogenase; and

[0033] (h) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid dehydrogenase, an isobutyryl-CoA reductase (aldehyde forming), and a branched-chain alcohol dehydrogenase.

[0034] In some embodiments, the present invention provides a method for producing isobutanol that includes culturing any of the aforementioned non-naturally occurring microbial organisms under conditions and for a sufficient period of time to produce isobutanol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows pathways to isopropanol, «-butanol, and isobutanol from 4- hydroxybutyryl-CoA.

[0036] Figure 2A shows the pathways for fixation of C0 ₂ to alpha-ketoglutarate, succinate and succinyl-CoA using the reductive TCA cycle.

[0037] Figure 2B shows exemplary pathways for the biosynthesis of isobutanol intermediate 4-hydroxybutyryl-CoA from alpha-ketoglutarate, succinate and succinyl-CoA ; the enzymatic transformations shown are carried out by the following enzymes: A. Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), B. Succinyl-CoA reductase (aldehyde forming), C. 4-Hydroxybutyrate dehydrogenase, D. 4-Hydroxybutyrate kinase, E. Phosphotrans- 4-hydroxybutyrylase, F. Succinate reductase, G. Succinyl-CoA reductase (alcohol forming), H. 4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase [0038] Figure 3 A shows the pathways for fixation of C0 ₂ to acetyl-CoA and pyruvate using the reductive TCA cycle.

[0039] Figure 3B shows exemplary pathways for the biosynthesis of isobutanol from pyruvate and acetyl-CoA; the enzymatic transformations shown are carried out by the following enzymes: a) acetolactate synthase, b) acetohydroxy acid isomeroreductase, c) acetohydroxy acid dehydratase, d) branched-chain keto acid decarboxylase, e) branched-chain alcohol

dehydrogenase, f) branched-chain keto acid dehydrogenase, g) isobutyryl-CoA reductase (aldehyde forming), h) valine dehydrogenase or transaminase, i) valine decarboxylase, j) omega transaminase, k) isobutyryl-CoA mutase, 1) acetoacetyl-CoA thiolase, m) 3-hydroxybutyryl-CoA dehydrogenase, n) crotonase, o) crotonyl-CoA reductase (butyryl-CoA forming).

[0040] Figure 4 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 1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng).

[0041] Figure 5 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.

[0042] Figure 6A shows the nucleotide sequence (SEQ ID NO: 1) of carboxylic acid reductase from Nocardia iowensis (GNM 720), and Figure 6B shows the encoded amino acid sequence (SEQ ID NO:2).

[0043] Figure 7A shows the nucleotide sequence (SEQ ID NO:3) of phosphpantetheine transferase, which was codon optimized, and Figure 7B shows the encoded amino acid sequence (SEQ ID NO:4).

[0044] Figure 8A shows the nucleotide sequence (SEQ ID NO:5) of carboxylic acid reductase from Mycobacterium smegmatis mc(2)155 (designated 890), and Figure 8B shows the encoded amino acid sequence (SEQ ID NO:6). [0045] Figure 9A shows the nucleotide sequence (SEQ ID NO:7) of carboxylic acid reductase from Mycobacterium avium subspecies paratuberculosis K-10 (designated 891), and Figure 9B shows the encoded amino acid sequence (SEQ ID NO:8).

[0046] Figure 10A shows the nucleotide sequence (SEQ ID NO: 9) of carboxylic acid reductase from Mycobacterium marinum M (designated 892), and Figure 10B shows the encoded amino acid sequence (SEQ ID NO: 10).

[0047] Figure 11 A shows the nucleotide sequence (SEQ ID NO: 11) of carboxylic acid reductase designated 891GA, and Figure 1 IB shows the encoded amino acid sequence (SEQ ID NO: 12).

DETAILED DESCRIPTION

[0048] This invention is directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze isopropanol, «-butanol, or isobutanol production. Pathways for the production of isopropanol, «-butanol, or isobutanol disclosed herein are based on 4-hydroxybutyryl-CoA as a starting material as shown in Figure 1. Successfully engineering these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation.

[0049] Although 4-hydroxybutyryl-CoA is not a highly common central metabolite, methods for engineering strains that synthesize 4-hydroxybutyryl-CoA have been described previously by Applicants in U.S. Patent Application No. 2009/0075351 , which is incorporated by reference herein in its entirety. An exemplary method involves synthesizing 4-hydroxybutyryl-CoA from succinyl-CoA by employing genes encoding succinic semialdehyde dehydrogenase (CoA- dependent), 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase, and

phosphotransbutyrylase activities.

[0050] Assuming glucose as the carbohydrate feedstock, synthesizing isopropanol from 4- hydroxybutyryl-CoA has a theoretical yield of 1.33 mol/mol. This is 33% higher than the maximum yield attainable using the pathway described by Subbian, et al. in U.S. 2008/0293125. Another benefit of the 4-hydroxybutyryl-CoA to isopropanol pathway is that it can enable redox balance in the absence of an external electron acceptor, whereas the pathway described in Subbian, et al. produces a surplus of NADH that must be dissipated by transferring electrons to an external electron acceptor.

[0051 ] Another compound that can be synthesized from 4-hydroxybutyryl-CoA is w-butanol. Assuming glucose as a carbohydrate feedstock, this pathway has a theoretical yield of about 1.00 mol mol yield of w-butanol. This yield is comparable to a route to w-butanol, native to many Clostridial species, that involves the formation of acetoacetyl-CoA from acetyl-CoA, followed by four reductions and a dehydration (Jones et al., Microbiol. Rev., 50:484-524 (1986)). A benefit of the present invention is that it bypasses the first three steps of this traditional butanol production pathway (i.e., acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, and crotonase) which form one molecule of crotonoyl-CoA from two acetyl-CoA molecules. Any or all of these enzymes represent potential bottlenecks to production. For example, although recombinant strains of E. coli have been engineered to express the requisite Clostridial genes for the traditional butanol synthetic pathway, titers of less than 1 g/L were reported (Atsumi, et al, Metabolic Engineering, 2008, 10, 305-311). Further benefits of the current invention are found in the underlying central metabolism required to produce w-butanol from 4- hydroxybutyryl-CoA as opposed to producing w-butanol from two acetyl-CoA molecules.

Specifically, to establish the highest yield and redox balance under anaerobic conditions, the traditional Clostridial route requires that two reducing equivalents per w-butanol are extracted from the conversion of pyruvate to acetyl-CoA (i.e., two pyruvate molecules must be oxidized to two acetyl-CoA molecules per butanol produced). This represents a challenge in organisms such as Escherichia coli, in which pyruvate dehydrogenase is not naturally highly active under anaerobic or microaerobic conditions. The production pathway disclosed herein needs only one pyruvate to be oxidized to acetyl-CoA per w-butanol produced. The additional reducing equivalent is generated by the conversion of isocitrate to alpha-ketoglutarate by isocitrate dehydrogenase.

[0052] Similarly, the theoretical yield of isobutanol via the 4-hydroxybutyryl-CoA pathway is about 1.00 mol/mol assuming glucose as the feedstock. One benefit of the current invention is that it bypasses the acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, and branched chain alpha-keto acid dehydrogenase steps of the conversion pathway from pyruvate to isobutyryl-CoA to isobutanol described in Donaldson et al., U.S. 20070092957. Alternatively, it bypasses the acetyl-CoA acetyltransferase, 3-hydroxybutyryl- CoA dehydrogenase, and crotonase steps required for the production of butyryl-CoA and isobutanol via another biosynthetic pathway also described in Donaldson, et al. Any or all of these enzymatic steps bypassed by the current invention represent potential bottlenecks to isobutanol production. In one embodiment of the present invention, the butyryl-CoA

intermediate is bypassed completely by converting 4-hydroxybutyryl-CoA to 3- hydroxyisobutyryl-CoA, which is subsequently dehydrated and reduced to isobutyryl-CoA.

[0053] Finally, this invention is also directed, in part, to methods for producing isopropanol, «-butanol, or isobutanol through culturing of these non-naturally occurring microbial organisms. Thus, any of the strains disclosed herein can be cultured under appropriate conditions, for a sufficient period of time to provide the commodity chemicals isopropanol, «-butanol, or isobutanol.

[0054] 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 an isopropanol, «-butanol, or isobutanol biosynthetic pathway.

[0055] 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. [0056] 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.

[0057] 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.

[0058] As used herein, the term "Co A" 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.

[0059] 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.

[0060] "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

[0061 ] 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 [0062] The non-naturally occurring microbial 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.

[0063] 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.

[0064] 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.

[0065] 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' exonuc lease 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.

[0066] 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 indicating 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. [0067] 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.

[0068] Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having isopropanol, «-butanol, or isobutanol 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.

[0069] 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.

[0070] 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: 1 1; 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.

[0071] In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having an isopropanol pathway having at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol. The isopropanol pathway includes an enzyme selected from the group consisting of a 4-hydroxybutyryl-CoA dehydratase, a crotonase, a 3- hydroxybutyryl-CoA dehydrogenase, an acetoacetyl-CoA synthetase, an acetyl- CoA:acetoacetate-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetate

decarboxylase, and an acetone reductase.

[0072] In some embodiments, the microbial organism includes two exogenous nucleic acids, each encoding an isopropanol pathway enzyme, while in other embodiments the microbial organism includes three exogenous nucleic acids, each encoding an isopropanol pathway enzyme. In some embodiments, the microbial organism includes four exogenous nucleic acids, each encoding an isopropanol pathway enzyme. In further embodiments, the microbial organism includes five exogenous nucleic acids, each encoding an isopropanol pathway enzyme. In yet further embodiments, the microbial organism includes six exogenous nucleic acids, each encoding an isopropanol pathway enzyme. The microbial organism can also include seven exogenous nucleic acids, each encoding an isopropanol pathway enzyme. Finally, the microbial organism can include eight exogenous nucleic acids, each encoding an isopropanol pathway enzyme. Any of the aforementioned genes that are inserted into the host organism can be a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

[0073] In accordance with the foregoing, the present invention provides a 4-hydroxybutyryl- CoA to isopropanol pathway that provides a nucleic acid encoding an enzyme that carries out the dehydration of 4-hydroxybutyryl-CoA to form crotonoyl-CoA as shown in step A of Figure 1. Crotonase subsequently hydrates crotonoyl-CoA to 3-hydroxybutyryl-CoA (step B) which, in turn, is oxidized to acetoacetyl-CoA by 3-hydroxybutyryl-CoA dehydrogenase (step C).

Acetoacetyl-CoA is converted to acetoacetate by a synthetase, transferase, or hydrolase (steps D, E, or F). The final two steps involve the decarboxylation of acetoacetate to form acetone (step G) and its subsequent reduction to isopropanol (step H).

[0074] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having an isopropanol 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 4- hydroxybutyryl-CoA to crotonoyl-CoA, crotonoyl-CoA to 3-hydroxybutyryl-CoA, 3- hydroxybutyryl-CoA to acetoacetyl-CoA, acetoacetacetyl-CoA to acetoacetate, acetoacetate to acetone, and acetone to isopropanol.

[0075] In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a «-butanol pathway having at least one exogenous nucleic acid encoding a «-butanol pathway enzyme expressed in a sufficient amount to produce «-butanol. The «-butanol pathway includes an enzyme selected from the group consisting of a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, a butyryl- CoA reductase (aldehyde forming), a butyraldehyde reductase, and a butyryl-CoA reductase (alcohol forming).

[0076] In some embodiments, the microbial organism includes two exogenous nucleic acids, each encoding an «-butanol pathway enzyme, while in other embodiments, the microbial organism includes three exogenous nucleic acids, each encoding an «-butanol pathway enzyme. In further embodiments, the microbial organism includes four exogenous nucleic acids, each encoding an «-butanol pathway enzyme. Any of the aforementioned nucleic acids can be provided as a heterologous nucleic acid. Such non-naturally occurring microbial organism can be grown in a substantially anaerobic culture medium.

[0077] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a «-butanol 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 4-hydroxybutyryl-CoA to crotonoyl-CoA, crotonoyl-CoA to butyryl-CoA, butyryl-CoA to «-butanol, butyryl-CoA to butyraldehyde, and butyraldehyde to «-butanol.

[0078] The 4-hydroxybutyryl-CoA to «-butanol pathway begins with the dehydration of 4- hydroxybutyryl-CoA to crotonoyl-CoA as shown in step A of Figure 1 , which is then reduced to butyryl-CoA (step I). Butyryl-CoA then undergoes two reductions carried out either by two separate enzymes, steps J and K, or a single dual-function enzyme as shown in step L.

[0079] In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having an isobutanol pathway having at least one exogenous nucleic acid encoding an isobutanol pathway enzyme expressed in a sufficient amount to produce isobutanol. The isobutanol pathway includes an enzyme selected from the group consisting of a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl-CoA mutase, a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, an isobutyryl-CoA reductase (aldehyde forming), an isobutyraldehyde reductase, and an isobutyryl-CoA reductase (alcohol forming). [0080] In some embodiments, the microbial organism includes two exogenous nucleic acids, each encoding an isobutanol pathway enzyme, while in other embodiments, the microbial organism includes three exogenous nucleic acids, each encoding an isobutanol pathway enzyme. In other embodiments, the microbial organism includes four exogenous nucleic acids, each encoding an isobutanol pathway enzyme. In still further embodiments, the microbial organism includes five exogenous nucleic acids, each encoding an isobutanol pathway enzyme. Any of the exogenous nucleic acid can be a heterologous nucleic acid. Such non-naturally occurring microbial organism can be grown in a substantially anaerobic culture medium.

[0081] In some embodiments, the non-naturally occurring organism has a set of isobutanol pathway enzymes that includes a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl-CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase. Such organisms can have one, two three, four, five, up to all nucleic acids encoding isobutanol pathway enzymes provided as exogenous nucleic acids.

[0082] In some embodiments, the non-naturally occurring organism has a set of isobutanol pathway enzymes comprises a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl-CoA mutase, and an isobutyryl-CoA reductase (alcohol forming). Such organisms can have one, two three, four, up to all nucleic acids encoding isobutanol pathway enzymes provided as exogenous nucleic acids.

[0083] In some embodiments, the non-naturally occurring organism has a set of isobutanol pathway enzymes comprises a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase. Such organisms can have one, two three, four, five, up to all nucleic acids encoding isobutanol pathway enzymes provided as exogenous nucleic acids.

[0084] In some embodiments, the non-naturally occurring organism has a set of isobutanol pathway enzymes comprises a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, and an isobutyryl-CoA reductase (alcohol forming). Such organisms can have one, two three, four, up to all nucleic acids encoding isobutanol pathway enzymes provided as exogenous nucleic acids. [0085] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having an isobutanol 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 4- hydroxybutyryl-CoA to crotonoyl-CoA, crotonoyl-CoA to butyryl-CoA, butyryl-CoA to isobutyryl-CoA, 4-hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA, 3-hydroxyisobutyryl-CoA to methacrylyl-CoA, methacrylyl-CoA to isobutyryl-CoA, isobutyryl-CoA to isobutanol, isobutyryl-CoA to isobutyraldehyde, and isobutryaldehyde to isobutanol.

[0086] The conversion of 4-hydroxybutyryl-CoA to isobutanol involves the formation of an isobutyryl-CoA intermediate. Isobutyryl-CoA can be formed from 4-hydroxybutyryl-CoA via a dehydration, reduction, and carbon backbone rearrangement as shown in steps A, I, and M, of Figure 1. Alternatively, this intermediate can be obtained via first carbon backbone

rearrangement, then dehydration and reduction as shown in steps Q, R, and S of Figure 1. Isobutyryl-CoA then undergoes two reductions to form isobutanol. The reductions are carried out either by two enzymes, steps N and O or a single dual- function enzyme as shown in step P.

[0087] In some embodiments, a non-naturally occurring microbial organism includes a microbial organism having an isobutanol pathway that includes at least one exogenous nucleic acid encoding a isobutanol pathway enzyme expressed in a sufficient amount to produce isobutanol; the non-naturally occurring microbial organism further includes:

[0088] (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha- ketoglutarate: ferredoxin oxidoreductase;

[0089] (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said 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 [0090] (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an ¾ hydrogenase, and combinations thereof;

[0091 ] wherein when the non-naturally occurring microbial organism includes an isobutanol pathway that converts 4-hydroxybutryl-CoA to isobutanol, a 4-hydroxybutyryl pathway is selected from:

[0092] (I) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (aldehyde forming), a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[0093] (II) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (alcohol forming), a 4-Hydroxybutyrate kinase, a

Phosphotrans-4-hydroxybutyrylase;

[0094] (III) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (aldehyde forming), a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[0095] (IV) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (alcohol forming), a 4-Hydroxybutyryl-CoA transferase, or 4- Hydroxybutyryl-CoA synthetase;

[0096] (V) an Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase), a 4-Hydroxybutyrate dehydrogenase, a 4-Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[0097] (VI) an Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase), a 4-Hydroxybutyrate dehydrogenase, a 4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[0098] (VII) a Succinate reductase, a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase; [0099] (VIII) a Succinate reductase, a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[00100] wherein the isobutanol pathway includes a pathway selected from:

[00101] (a) a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl- CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase;

[00102] (b) a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl- CoA mutase, and an isobutyryl-CoA reductase (alcohol forming);

[00103] (c) a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase;

[00104] (d) a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, and an isobutyryl-CoA reductase (alcohol forming);

[00105] (e) an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA reductase (butyryl-CoA forming) an isobutyryl-CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and a branched-chain alcohol dehydrogenase;

[00106] (f) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase;

[00107] (g) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a valine dehydrogenase or transaminase, a valine decarboxylase, an omega transaminase, and a branched-chain alcohol dehydrogenase; and

[00108] (h) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid dehydrogenase, an isobutyryl-CoA reductase (aldehyde forming), and a branched-chain alcohol dehydrogenase. [00109] In some embodiments, non-naturally occurring microbial organism (e.g., having pathway (i)) further includes 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, ferredoxin, and combinations thereof.

[001 10] In some embodiments, non-naturally occurring microbial organism (e.g., having pathway (ii)) further includes 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, and combinations thereof.

[001 1 1] In some embodiments, the non-naturally occurring microbial organism includes two, three, four, five, six, or seven, eight, nine, or ten exogenous nucleic acids each encoding an isobutanol pathway enzyme.

[001 12] In some embodiments, the non-naturally occurring microbial organism (e.g., having pathway (i)) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

[001 13] In some embodiments, the non-naturally occurring microbial organism (e.g., having pathway (ii)) comprises two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

[001 14] In some embodiments, the non-naturally occurring microbial organism has at least one exogenous nucleic acid that is a heterologous nucleic acid.

[001 15] In some embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

[001 16] Also provided herein is a non-naturally occurring microbial organism having an isobutanol pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a isobutanol pathway enzyme expressed in a sufficient amount to produce isobutanol; said non-naturally occurring microbial organism further comprising: [00117] (i) a reductive TCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme selected from the group consisting of an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha- ketoglutarate: ferredoxin oxidoreductase;

[001 18] (ii) a reductive TCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme selected from the group consisting of a pyruvate: ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an ¾ hydrogenase; or

[001 19] (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an ¾ hydrogenase, and combinations thereof;

[00120] wherein said microbial organism comprises an isobutanol pathway that converts 4- hydroxybutyryl-CoA to isobutanol,

[00121] and wherein said microbial organism further comprises a pathway selected from the group consisting of:

[00122] (a) a Succinyl-CoA transferase, Succinyl-CoA synthetase or succinyl-CoA ligase; a Succinyl-CoA reductase (aldehyde forming); a 4-Hydroxybutyrate dehydrogenase; a 4- Hydroxybutyrate kinase; and a Phosphotrans-4-hydroxybutyrylase;

[00123] (b) a Succinyl-CoA transferase, Succinyl-CoA synthetase or succinyl-CoA ligase); a Succinyl-CoA reductase (alcohol forming); a 4-Hydroxybutyrate kinase; and a Phosphotrans-4- hydroxybutyrylase;

[00124] (c) a Succinyl-CoA transferase, Succinyl-CoA synthetase or succinyl-CoA ligase); a Succinyl-CoA reductase (aldehyde forming); a 4-Hydroxybutyrate dehydrogenase; and a 4- Hydroxybutyryl-CoA transferase or 4-Hydroxybutyryl-CoA synthetase;

[00125] (d) a Succinyl-CoA transferase, Succinyl-CoA synthetase or succinyl-CoA ligase; a Succinyl-CoA reductase (alcohol forming); and a 4-Hydroxybutyryl-CoA transferase or 4- Hydroxybutyryl-CoA synthetase; [00126] (e) an Alpha-ketoglutarate decarboxylase or (a Glutamate dehydrogenase and/or Glutamate transaminase; a Glutamate decarboxylase; a 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase); a 4-Hydroxybutyrate dehydrogenase; a 4-Hydroxybutyrate kinase; and a Phosphotrans-4-hydroxybutyrylase;

[00127] (f) an Alpha-ketoglutarate decarboxylase or (a Glutamate dehydrogenase and/or Glutamate transaminase; a Glutamate decarboxylase; a 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase); a 4-Hydroxybutyrate dehydrogenase; and a 4-Hydroxybutyryl- CoA transferase or 4-Hydroxybutyryl-CoA synthetase;

[00128] (g) a Succinate reductase; a 4-Hydroxybutyrate dehydrogenase; a 4-Hydroxybutyrate kinase; and a Phosphotrans-4-hydroxybutyrylase; and

[00129] (h) a Succinate reductase; a 4-Hydroxybutyrate dehydrogenase; and a 4- Hydroxybutyryl-CoA transferase or 4-Hydroxybutyryl-CoA synthetase;

[00130] and wherein said isopropanol pathway is selected from the group consisting of:

[00131] (A) a 4-hydroxybutyryl-CoA dehydratase; a crotonoyl-CoA reductase; an isobutyryl-CoA mutase; an isobutyryl-CoA reductase (aldehyde forming); and an

isobutyraldehyde reductase;

[00132] (B) a 4-hydroxybutyryl-CoA dehydratase; a crotonoyl-CoA reductase; an isobutyryl- CoA mutase; and an isobutyryl-CoA reductase (alcohol forming);

[00133] (C) a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA

dehydratase; a methacrylyl-CoA-reductase; an isobutyryl-CoA reductase (aldehyde forming); and an isobutyraldehyde reductase;

[00134] (D) a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA

dehydratase; a methacrylyl-CoA-reductase; and an isobutyryl-CoA reductase (alcohol forming);

[00135] (E) an acetoacetyl-CoA thiolase; a 3-hydroxybutyryl-CoA dehydrogenase; a crotonase; a crotonyl-CoA reductase (butyryl-CoA forming); an isobutyryl-CoA mutase; an isobutyryl-CoA reductase (aldehyde forming); and a branched-chain alcohol dehydrogenase; [00136] (F) an acetolactate synthase; an acetohydroxy acid isomeroreductase; an acetohydroxy acid dehydratase; a branched-chain keto acid decarboxylase; and a branched-chain alcohol dehydrogenase;

[00137] (G) an acetolactate synthase; an acetohydroxy acid isomeroreductase; an acetohydroxy acid dehydratase; a valine dehydrogenase or transaminase; a valine decarboxylase; an omega transaminase; and a branched-chain alcohol dehydrogenase; and

[00138] (H) an acetolactate synthase; an acetohydroxy acid isomeroreductase; an acetohydroxy acid dehydratase; a branched-chain keto acid dehydrogenase; an isobutyryl-CoA reductase (aldehyde forming); and a branched-chain alcohol dehydrogenase.

[00139] In some embodiments, the non-naturally occurring microbial organism comprising

(i) , further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of 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, ferredoxin, and combinations thereof.

[00140] In some embodiments, the non-naturally occurring microbial organism comprising

(ii) , further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl- CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

[00141] In some embodiments, the microbial organism comprises two, three, four, five, six, or seven, eight, nine, or ten exogenous nucleic acids, each encoding an isobutanol pathway enzyme.

[00142] In some embodiments, the microbial organism comprising (i) comprises two, three or four exogenous nucleic acids, each encoding a reductive TCA pathway enzyme.

[00143] In some embodiments, the microbial organism comprising (ii) comprises two, three or four exogenous nucleic acids, each encoding a reductive TCA pathway enzyme.

[00144] In some embodiments, the at least one exogenous nucleic acid is a heterologous nucleic acid. [00145] In some embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

[00146] In certain embodiments, the microbial organism comprises a nucleic acid encoding each of the enzymes in the recited pathway.

[00147] Also provided herein are methods for producing isobutanol, comprising culturing a non-naturally occurring microbial organism provided herein under conditions and for a sufficient period of time to produce isobutanol. In some embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

[00148] In accordance with embodiments of the invention, the following gene candidates encoding isobutanol pathway enzymes are applicable to the production of isobutanol in a non- naturally occurring microbial organism of the invention. Specifically, the following candidate genes encode enzymes useful in carrying out isobutanol synthesis as shown in Figure 3.

[00149] The terms "acetolactate synthase" and "acetolactate synthetase" are used

interchangeably herein to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and C0 ₂ . Preferred acetolactate synthases are known by the EC number 2.2.1.6 ( Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB 15618, Z99122, NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), Klebsiella pneumoniae (GenBank Nos: AAA25079,

M73842), and Lactococcus lactis (GenBank Nos: AAA25161 , L16975).

[00150] The terms "acetohydroxy acid isomeroreductase" and "acetohydroxy acid

reductoisomerase" are used interchangeably herein to refer to an enzyme that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate using NADPH (reduced nicotinamide adenine dinucleotide phosphate) as an electron donor. Preferred acetohydroxy acid

isomeroreductases are known by the EC number 1.1.1.86 and sequences are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos:

NP 418222, NC 000913), Saccharomyces cerevisiae (GenBank Nos: NP 013459, NC 001 144, Methanococcus maripaludis (GenBank os: CAF30210, BX957220), and Bacillus, subtilis (GenBank Nos: CAB14789, Z99118).

[00151] The term "acetohydroxy acid dehydratase" refers to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate. Preferred acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. These enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP 026248, NC 000913), S. cerevisiae (GenBank Nos: NP 012550, NC 001142, M. maripaludis (GenBank Nos: CAF29874, BX957219), and B. subtilis (GenBank Nos: CAB14105, Z99115).

[00152] The term "branched-chain a-keto acid decarboxylase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to isobutyraldehyde and CO 2 . Preferred branched- chain a-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166, AY548760; CAG34226, AJ746364, Salmonella typhimurium (GenBank Nos: NP 461346, NC 003197), and Clostridium acetobutylicum (GenBank Nos: NP 149189, NC 001988).

[00153] The term "branched-chain alcohol dehydrogenase" refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Preferred branched-chain alcohol

dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH (reduced nicotinamide adenine dinucleotide) and/or NADPH as electron donor and are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos:

NP 010656, NC 001136; NP 014051 NC 001 145), E. coli (GenBank Nos: NP 417484, NC 000913), and C. acetobutylicum (GenBank Nos: NP 349892, NC 003030; NP 349891 , NC 003030).

[00154] The term "branched-chain keto acid dehydrogenase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), using NAD + (nicotinamide adenine dinucleotide) as electron acceptor. Preferred branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. These branched-chain keto acid dehydrogenases are comprised of four subunits and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, B. subtilis (GenBank Nos: CAB14336, Z99116; CAB14335, Z991 16; CAB14334, Z991 16; and CAB14337, Z991 16) and Pseudomonas putida (GenBank os: AAA65614, M57613; AAA65615, M57613; AAA65617, M57613; and AAA65618, M57613).

[00155] The term "acylating aldehyde dehydrogenase" refers to an enzyme that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, using either NADH or NADPH as electron donor. Preferred acylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. These enzymes are available from multiple sources, including, but not limited to, Clostridium beijerinckii (GenBank Nos: AAD31841, AF157306), C. acetobutyhcum (GenBank Nos: NP 149325, NC 001988; NP 149199, NC 001988), P. putida (GenBank Nos:

AAA89106, U13232), and Thermus thermophilus (GenBank Nos: YP 145486, NC 006461).

[00156] The term "transaminase" refers to an enzyme that catalyzes the conversion of a- ketoisovalerate to L-valine, using either alanine or glutamate as amine donor. Preferred transaminases are known by the EC numbers 2.6.1.42 and 2.6.1.66. These enzymes are available from a number of sources. Examples of sources for alanine-dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026231, NC 000913) and Bacillus lichenifonnis (GenBank Nos: YP 093743, NC 006322). Examples of sources for glutamate- dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026247, NC 000913), S. cerevisiae (GenBank Nos: NP 012682, NC 001142) and Methanobacterium

thermoautotrophicum (GenBank Nos: NP 276546, NC 000916).

[00157] The term "valine dehydrogenase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, using NAD(P)H as electron donor and ammonia as amine donor. Preferred valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP 628270, NC 003888) and B. subtilis (GenBank Nos: CAB14339, Z99116).

[00158] The term "valine decarboxylase" refers to an enzyme that catalyzes the conversion of L-valine to isobutylamine and CO 2 . Preferred valine decarboxylases are known by the EC number 4.1.1.14. These enzymes are found in Streptomycetes, such as for example,

Streptomyces viridifaciens (GenBank Nos: AAN10242, AY116644). [00159] The term "omega transaminase" refers to an enzyme that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as amine donor. Preferred omega transaminases are known by the EC number 2.6.1.18 and are available from a number of sources, including, but not limited to, Alcaligenes denitrificans (AAP92672, AY330220), Ralstonia eutropha (GenBank Nos: YP 294474, NC 007347), Shewanella oneidensis (GenBank Nos: NP 719046, NC 004347), and P. putida (GenBank Nos: AAN66223, AE016776).

[00160] The term "isobutyryl-CoA mutase" refers to an enzyme that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme B 12 as cofactor. Preferred isobutyryl-CoA mutases are known by the EC number 5.4.99.13. These enzymes are found in a number of Streptomycetes, including, but not limited to, Streptomyces cinnamonensis (GenBank Nos: AAC08713, U67612; CAB59633, AJ246005), S. coelicolor (GenBank Nos: CAB70645, AL939123; CAB92663, AL939121), and Streptomyces avermitilis (GenBank Nos: NP 824008, NC 003155; NP 824637, NC 003155).

[00161] Exemplary genes for the acetoacetyl-CoA thiolase step include atoB which can catalyze the reversible condensation of 2 acetyl-CoA molecules (Sato et al., supra, 2007), and its horn olog yqeF. Non- E. coli genes that can be used include phaA from R. eutropha (Jenkins, L. S. and W. D. Nunn. Journal of Bacteriology 169:42-52 (1987)), and the two ketothiolases, thiA and MB, from Clostridium acetobutylicum (Winzer et al., Journal of Molecular Microbiology and Biotechnology 2:531-541 (2000)). The sequences for these genes can be found at the following Genbank accession numbers: atoB NP 416728.1 Escherichia coli yqeF NP 417321.2 Escherichia coli phaA YP 725941 Ralstonia eutropha thiA NP 349476.1 Clostridium acetobutylicum thiB NP 149242.1 Clostridium acetobutylicum

[00162] An exemplary gene from E. coli which can be used for conferring 3-hydroxybutyryl- CoA dehydrogenase transformation activity is paaH (Ismail et al., European Journal of Biochemistry 270:3047-3054 (2003)). on E. coli genes applicable for conferring this activity include AA072312.1 from E. gracilis (W inkier et al, Plant Physiology 131 :753-762 (2003)), paaC from Pseudomonas putida (Olivera et al, PNAS USA 95:6419-6424 (1998)), paaC from Pseudomonas fluorescens (Di Gennaro et al., Archives of Microbiology 188: 1 17-125 (2007)), and hbd from C. acetobutylicum (Atsumi et al., Metabolic Engineering (2007) and Boynton et al., Journal of Bacteriology 178:3015-3024 (1996)). The sequences for each of these exemplary genes can be found at the following Genbank accession numbers: paaH P 415913.1 Escherichia coli

AA072312.1 Euglena gracilis paaC NP 745425.1 Pseudomonas putida paaC ABF82235.1 Pseudomonas fluorescens hbd P 349314.1 Clostridium acetobutylicum

[00163] Exemplary genes encoding the crotonase step include, for example, maoC (Park and Lee, Journal Bacteriology 185:5391 -5397 (2003)), paaF (Ismail et al., European Journal of Biochemistry 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol. 113-116:335- 346 (2004) and Park and Yup, Biotechnol. Bioeng. 86:681-686 (2004)), and paaG (Ismail et al, European Journal of Biochemistry 270:3047-3054 (2003); Park and Lee, Appl. Biochem.

Biotechnol. 1 13-1 16:335-346 (2004) and Park and Yup, Biotechnol. Bioeng. 86:681-686 (2004)). Other genes which can be used to produce the gene product catalyzing this step , for example, paaA,paaB, and paaN from P. putida (Olivera et al, PNAS USA 95:6419-6424 (1998)) and P. fluorescens (Di Gennaro et al., Archives of Microbiology 188: 1 17-125 (2007)). The gene product of crt from C. acetobutylicum also can be used (Atsumi et al., Metabolic Engineering (2007) and Boynton et al., Journal of Bacteriology 178: 3015-3024 (1996. The sequences for each of these exemplary genes can be found at the following Genbank accession numbers: maoC NP 415905.1 Escherichia coli paaF NP 415911.1 Escherichia coli paaG NP 415912.1 Escherichia coli paaA P 745427.1 P seudomonas putida paaA ABF82233.1 P seudomonas fluorescens paaB NP 745426.1 P seudomonas putida paaB ABF82234.1 P seudomonas fluorescens paaN NP 745413.1 P seudomonas putida paaN ABF82246.1 P seudomonas fluorescens crt NP 349318.1 Clostridium acetobutylicum

[00164] An exemplary gene which can be introduced into a non-naturally occurring microbial organism of the invention to confer crotonyl-CoA reductase (butyryl-CoA forming) activity is the mitochondrial enoyl-CoA reductase from E. gracilis Hoffmeister et al., supra (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence has been cloned and expressed in E. coli. This approach for heterologous expression of membrane targeted polypeptides in a soluble form is well known to those skilled in the art of expressing eukaryotic genes, particularly those with leader sequences that may target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597, from the prokaryote Treponema denticola represents also can be employed to confer enoyl-CoA reductase activity (Tucci and Martin, FEBS Letters 581 : 1561-1566 (2007)). Butyryl-CoA dehydrogenase, encoded by bed from C. acetobutylicum, is a further exemplary enzyme that can be used to confer enoyl-CoA reductase activity onto a host microbial organism of the invention (Atsumi et al., Metabolic Engineering (2007) and Boynton et al., Journal of Bacteriology 178: 3015-3024 (1996)). Alternatively, E. coli genes exhibiting this activity can be obtained using methods well known in the art (see, for example, Mizugaki et al., Chemical & Pharmaceutical Bulletin 30:206-213 (1982) and Nishimaki et al., Journal of Biochemistry 95: 1315-1321 (1984)). The sequences for each of the above exemplary genes can be found at the following Genbank accession numbers: TER Q5EU90.1 Euglena gracilis

TDE0597 NP 971211.1 Treponema denticola bcd P 349317.1 Clostridium acetobutylicum

[00165] At least three mitochondrial enoyl-CoA reductase enzymes exist in E. gracilis that similarly are applicable for use in the invention. Each enoyl-CoA reductase enzyme exhibits a unique chain length preference (Inui et al., European Journal of Biochemistry 142: 121-126 (1984)), which is particularly useful for dictating the chain length of the desired primary alcohol products of the invention. EST's ELL00002199, ELL00002335, and ELL00002648, which are all annotated as mitochondrial trans-2-enoyl-CoA reductases, can be used to isolate these additional enoyl-CoA reductase genes as described further below.

[00166] This invention is also directed, in part to engineered biosynthetic pathways to improve carbon flux through a central metabolism intermediate en route to isobutanol. 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 isobutanol. 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.

[00167] 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 isobutanol 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 limited to, the atmosphere, either as found in nature or generated.

[00168] 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 2a). 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.

[00169] 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.

[00170] 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, thioredoxins, and reduced flavodoxins. 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-ketoglutar ate: 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.

[00171] The reductive TCA cycle was first reported in the green sulfur photosynthetic bacterium Chlorobium Umicola (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, FEMS Microbiol. Rev. 28:335-352 (2004)).

[00172] 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.

[00173] 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 [00174] 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, 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. Enzymes and the corresponding genes required for these activities are described herein above.

[00175] 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 isobutanol 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.

[00176] In some embodiments, a isobutanol 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.

[00177] In some embodiments a non-naturally occurring microbial organism having an isobutanol 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 fumarate reductase, an isocitrate dehydrogenase, an 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 .

[00178] In some embodiments a method includes culturing a non-naturally occurring microbial organism having a isobutanol 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 fumarate reductase, an isocitrate

dehydrogenase, an 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.

[00179] In some embodiments a non-naturally occurring microbial organism having an isobutanol 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 fumarate reductase, a pyruvate: ferredoxin oxidoreductase, an isocitrate dehydrogenase, sn aconitase, and an alpha-ketoglutarate: ferredoxin oxidoreductase.

[00180] In some embodiments a non-naturally occurring microbial organism having an isobutanol 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 isobutanol. [00181] In some embodiments, the non-naturally occurring microbial organism having an isobutanol pathway includes two exogenous nucleic acids, each encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism having an isobutanol 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, 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.

[00182] In some embodiments, the non-naturally occurring microbial organisms having an isobutanol 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.

[00183] In some embodiments, the non-naturally occurring microbial organism having an isobutanol 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.

[00184] In some embodiments, the non-naturally occurring microbial organism having an isobutanol 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 an isobutanol pathway utilizes hydrogen for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having an isobutanol pathway utilizes CO for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having an isobutanol pathway utilizes combinations of CO and hydrogen for reducing equivalents.

[00185] In some embodiments, the non-naturally occurring microbial organism having an isobutanol 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.

[00186] In some embodiments, the non-naturally occurring microbial organism having an isobutanol 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.

[00187] In some embodiments, the non-naturally occurring microbial organism having an isobutanol 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.

[00188] 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.

[00189] In an additional embodiment, the invention provides a non-naturally occurring microbial organism having an isobutanol 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 4- hydroxybutyryl-CoA to crotonyl-CoA, 4-hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA, crotonyl-CoA to butyryl-CoA, butyryl-CoA to isobutyryl-CoA, 3-hydroxyisobutyryl-CoA to methacrylyl-CoA, methacrylyl-CoA to isobutyryl-CoA, isobutyryl-CoA to isobutanol, isobutyryl-CoA to isobutyraldehyde, isobutyraldehyde to isobutanol, acetoacetyl-CoA to 3- hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to crotonyl-CoA, pyruvate to 2-hydroxy-2-methyl acetoacetate, 2-hydroxy-2-methyl acetoacetate to 2-keto-3-hydroxyisoamylate, 2-keto-3- hydroxyisoamylate to 2,3-dihydroxyisoamylate, 2-keto-3-hydroxyisoamylate to 2- ketoisoamylate, 2,3-dihydroxyisoamylate to 2-ketoisoamylate, 2-ketoisoamylate to 2-amino isoamylate, 2-aminoisoamylate to isobutylamine, isobutylamine to isobutyraldehyde, 2- ketoisoamylate to isobutyraldehyde, and 2-ketoisoamylate to isobutyryl-CoA. 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 isobutanol pathway, such as that shown in Figures 1 and 3B.

[00190] While generally described herein as a microbial organism that contains a isobutanol pathway, it is understood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding an isobutanol pathway enzyme expressed in a sufficient amount to produce an intermediate of an isobutanol pathway. For example, as disclosed herein, an isobutanol pathway is exemplified in Figure 1 , in conjunction with Figure 2, and 3A in conjunction with Figure 3B. Therefore, in addition to a microbial organism containing a isobutanol pathway that produces isobutanol, the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a isobutanol pathway enzyme, where the microbial organism produces a isobutanol pathway intermediate.

[00191] 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, 2, 3A B, 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 isobutanol pathway intermediate can be utilized to produce the intermediate as a desired product. [00192] Successfully engineering any of these pathways entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing fermentation conditions, and assaying for product formation following fermentation. To engineer a production host for the production of any of the aforementioned products, one or more exogenous DNA sequence(s) can be expressed in microorganisms. In addition, the microorganisms can have endogenous gene(s) functionally deleted. These modifications will enable the production of isopropanol, «-butanol, or isobutanol using renewable feedstocks.

[00193] Below, we describe a number of biochemically characterized genes capable of encoding enzymes that catalyze each of the steps shown in Figure 1. Although described in the context of an engineered E. coli, one skilled in the art can apply these teachings to essentially any other organism. Specifically, genes are listed that are native to E. coli as well as other genes in other organisms that can be applied to catalyze the appropriate transformations when properly cloned and expressed.

[00194] 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.

[00195] As disclosed herein, the isobutanol and other products proceed by way of

intermediates that are carboxylic acids (or derivatives of carboxylic acids which are readily converted to carboxylic acids). Such carboxylic acids 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 isobutyrate, ethyl isobutyrate, and n-propyl isobutyrate.

Other biosynthetically accessible O-carboxylates can include medium to long chain groups, that is C7-C22, O-carboxylate esters derived from fatty alcohols, such 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.

[00196] All transformations depicted in Figure 1 fall into the 10 general categories of transformations shown in Table 1. Below is described a number of biochemically characterized genes in each category. Specifically listed are genes that can be applied to catalyze the appropriate transformations in Figure 1 when properly cloned and expressed. Exemplary genes for each of the steps in Figure 1 are provided further below in Table 37.

[00197] Table 1 shows the enzyme types useful to convert common central metabolic intermediates into isopropanol, «-butanol, or isobutanol. The first three digits of each label correspond to the first three Enzyme Commission number digits which denote the general type of transformation independent of substrate specificity. Table 1

[00198] Two transformations in Figure 1 fall into the category of oxidoreductases that reduce an aldehyde to alcohol. Specifically, these are butyraldehyde reductase and isobutyraldehyde reductase, as shown in steps K and O, respectively.

[00199] Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include air A encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol, 66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al., Nature, 451 :86-89 (2008)), yqhD from E. coli which has preference for molecules longer than C3 (Sulzenbacher et al., J. of Molecular Biology, 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyraldehyde into butanol (Walter et al., J. of Bacteriology, 174:7149-7158 (1992)). The gene product of yqhD catalyzes the reduction of acetaldehyde, malondialdehyde, propionaldehyde, butyraldehyde, and acrolein using NADPH as the cofactor ( Perez et al., J Biol. Chem., 283:7346-7353 (2008)). ADHl from Zymomonas mobilis has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol, 22:249-254 (1985)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 2. Table 2

[00200] Enzymes exhibiting 3-hydroxybutyraldehyde reductase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha Bravo et al., J. Forensic Sci., 49:379-387(2004)), Clostridium kluyveri (Wolff et al, Protein Expr. Purif, 6:206-212(1995)) and Arabidops is thaliana ( Breitkreuz et al., J. Biol. Chem., 278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenase adhl from Geobacillus

thermoglucosidasius ( Jeon et al., J Biotechnol, 135: 127-133 (2008)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 3.

Table 3

[00201] Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase which catalyzes the reversible oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., J Mol Biol, 352:905-917 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate ( Manning et al., Biochem J, 231 :481-484 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens ( Hawes et al., Methods Enzymol , 324:218-228 (2000)) and Oryctolagus cuniculus (Howes et al, supra; Chowdhury et al., Biosci. Biotechnol Biochem. , 60:2043-2047 (1996)), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Hawes et al., supra; Chowdhury et al., Biosci. Biotechnol Biochem., 60:2043-2047 (1996); Chowdhury et al, Biosci. Biotechnol Biochem., 67:438-441(2003)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 4.

Table 4

[00202] Oxidoreductases that convert a ketone functionality to the corresponding hydroxyl group are exemplified by step H, for the conversion of acetone to isopropanol, and by step C for the conversion of 3-hydroxybutyryl-CoA to acetoacetyl-CoA as shown in Figure 1. An exemplary alcohol dehydrogenase that converts acetone to isopropanol was shown in C.

beijerinckii ( Ismaiel et al., J. Bacteriol, 175:5097-5105(1993)) and T. brockii (Lamed et al., Biochem. J, 195: 183-190(1981); Peretz et al, Biochemistry, 28:6549-6555 (1989)). The gene product of adhA from Pyrococcus furiosus, which exhibits maximum activity on 2-pentanol and pyruvaldehyde, was shown to have very broad specificity which includes isopropanol and acetone (van der et al., Eur J Biochem, 268:3062-3068 (2001)). Yet another secondary alcohol dehydrogenase with activity on isopropanol and acetone is encoded by the gene product of adh-A from Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng, 86:55-62 (2004)). These genes along with others are listed below in Table 5.

Table 5

[00203] 3-hydroxybutyryl-CoA dehydrogenase (Step C) catalyzing the formation of acetoacetyl-CoA from 3-hydroxybutyryl-CoA participates in the acetyl-CoA fermentation pathway to butyrate in several species of Clostridia and has been studied in detail (Jones and Woods, Microbiol. Rev. 50:484-524 (1986). The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., J. Bacteriol. 171 :6800-6807 (1989). Additionally, subunits of two fatty acid oxidation complexes in E. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock and Schulz, Meth. Enzymol. 71 Pt C, 403-411 (1981). Yet other genes demonstrated to catalyze this reversible transformation are phbB from Zoogloea ramigera (Ploux et al., Eur. J. Biochem. 174: 177-182 (1988) and phaB from Rhodobacter sphaeroides (Alber et al., Mol. Microbiol. 61 :297-309 (2006). The former gene is NADPH-dependent, its nucleotide sequence has been determined (Peoples and Sinskey, Mol. Microbiol. 3:349-257 (1989), and the gene has been expressed in E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Ploux et al., supra).

Additional genes include Hbdl (C-terminal domain) and Hbd2 (N-terminal domain) in

Clostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334: 12-23 (1974)) and HSD17B10 Bos taurus (Wakil et al, J. Biol. Chem. 207:631-638 (1954). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 6.

Table 6

[00204] A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007) as shown in Table 7.

Table 7

[00205] Alternatively, there exists several exemplary alcohol dehydrogenases that convert a ketone to a hydroxyl functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (IdhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on substrates of various chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate Steinbuchel et al., Eur. J. Biochem., 130:329-334 (1983)).

[00206] Transformations in Figure 1 also rely on the two-step reduction of acyl-CoA to the corresponding alcohol. For example, step L in the butanol pathway and step P in the isobutanol pathway rely on this transformation. Exemplary two-step oxidoreductases that convert an acyl- CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from £. coli (Kessler et al, FEES. Lett., 281 :59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., J. Bacteriol, 184:821-830 (2002)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA ( Kazahaya et al., J. Gen. Appl. Microbiol, 18:43-55 (2005)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 8. Table 8

[00207] Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH- dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., J. Bacteriol., 184:2404-2410 (2002); Strauss et al., Eur. J. Biochem., 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., supra). Enzymes in other organisms including Roseiflexus castenholzii,

Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 9.

Table 9

[00208] Longer chain acyl-CoA molecules can be reduced by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et al, Plant Physiology, 122:635-644 (2000)) (FAR, AAD38039.1, Simmondsia chinensis).

[00209] The pathways disclosed herein also involve oxidoreductase-type transformations that convert an acyl-CoA to an aldehyde. Specifically, Steps J and N catalyze the reduction of butytyl-CoA to butyraldehyde and isobutyryl-CoA to isobutyraldehyde respectively. Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde. Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase (Reiser et al., Journal of Bacteriology, 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Environ. Microbiol, 68: 1192-1 195 (2002)), and a CoA- and NADP- dependent succinate semialdehyde

dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling et al., J. Bacteriol 178:871-880 (1996)). SucD of P. gingivalis is another succinate semialdehyde dehydrogenase (Takahashi et al., J. Bacteriol 182:4704-4710 (2000)). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another enzyme demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al. J. Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al, Bacteriol Lett. 27:505-510 (2005)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 10.

Table 10

[00210] An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl- CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg et al., Science 318: 1782-1786 (2007); Thauer Science 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., supra; Hugler et al., J.

Bacteriol.184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., supra; Berg et al., supra). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli ((Alber et al., supra). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde. Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzymes have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional genes can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another enzyme for CoA- acylating aldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth et al., Appl. Environ Microbiol. 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., supra). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 11.

Table 11

[00211] Referring to Figure 1, step I refers to the conversion of crotonyl-CoA to butyryl-CoA by crotonyl-CoA reductase and step S refers to the conversion of methacryl-CoA to isobutyryl- CoA by methacrylyl-CoA reductase. Enoyl-CoA reductase enzymes are enzymes that can carry out either step. One exemplary enoyl-CoA reductase is the gene product of bed from C.

acetobutylicum (Boynton et al., J. Bacteriol. 178:3015-3024 (1996); Atsumi et al., Metab. Eng. (2007)), which naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. An additional enzyme for the enoyl-CoA reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this sequence following the removal of its mitochondrial targeting leader sequence was cloned in E. coli resulting in an active enzyme (Hoffmeister et al., supra). This approach is well known to those skilled in the art of expressing eukaryotic genes, particularly those with leader sequences that can target the gene product to a specific intracellular compartment, in prokaryotic organisms. A close homolog of this gene, TDE0597, from the prokaryote Treponema denticola represents a third enoyl-CoA reductase which has been cloned and expressed in E. coli (Tucci et al., FEBS Lett, 581 : 1561-1566 (2007)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 12.

Table 12

[00212] Isobutyryl-CoA dehydrogenase is another enzyme for step S of Figure 1, though it naturally catalyzes the oxidation of isobutyryl-CoA to methacrylyl-CoA. The crystal structure of the human isobutyryl-CoA dehydrogenase with and without the bound substrate has been determined (Battaile et al, J. Biol. Chem. 279: 16526-16534 (2004)). Additional isobutyryl-CoA dehydrogenases from Mus musculus and Rhodopseudomonas palustris can be inferred by sequence similarity. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 13.

Table 13

[00213] Yet further enzymes include 2-enoate reductases (EC 1.3.1.31) that are known to catalyze the NADH-dependent reduction of a wide variety of a, β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). 2-enoate reductase is encoded by enr in several species of Clostridia (Giesel, et al. Arch. Microbiol. 135:51-57 (1983)) including C. tyrobutyricum, and C. thermoaceticum (now called Moorella thermoaceticum) (Rohdich et al., supra). In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases have been reported, out of which one has been characterized (Seedorf al , Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008). The enr genes from both C. tyrobutyricum and C. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel et al., supra). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., supra). The C. thermoaceticum enr gene has also been expressed in an enzymatically active form in E. coli (Rohdich et al., supra). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 14.

Table 14

[00214] Step E of Figure 1 refers to the conversion of acetoacetyl-CoA to acetoacetate by acetyl-CoA:acetoacetate-CoA transferase or a similar transferase. The E. coli enzyme encoded by atoA (alpha subunit) and atoD (beta subunit) (Vanderwinkel et al., Biochem. Biophys. Res. Comm. 33:902-908 (1968)); Korolev et al, Acta Crystallagr. D Biol Crystallagr. 58:2116-2121 (2002)), also known as acetate-CoA transferase (EC 2.8.3.8), catalyzes this exact transformation and has additionally been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl. Environ. Microbiol. 58: 1435-1439 (1992)), valerate (Vanderwinkel supra) and butanoate (Vanderwinkel supra). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl.

Environ. Microbiol. 68:5186-5190 (2002)), Clostridium acetobutylicum (90), and Clostridium saccharoperbutylacetonicum (1 12). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 15

Table 15

[00215] Additional exemplary transferase transformations are catalyzed by the gene products of catl, cat2, and cat3 of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity, respectively (Aberthart et al., J. Chem. Soc. 6: 1404-1406 (1979); Agnihotri et al. Med. Chem. 11 :9-20 (2003)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 16.

Table 16

[00216] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., supra). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 17.

Table 17

[00217] Additional enzymes capable of converting acetoacetyl-CoA to acetoacetate include succinyl-CoA:3-ketoacid CoA transferases which utilize succinate as the CoA acceptor.

Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein. Expr. Purif. 53:396-403 (2007)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 18.

Table 18

[00218] Step F of Figure 1 refers to the conversion of acetoacetyl-CoA to acetoacetate by acetoacetyl-CoA hydrolase. Such activity has been detected in Rattus norvegicus (Patel et al., Biochem. J. Biochem. J. 176:951-958 (1978)), Bos taurus (Drummond et al., J. Biol. Chem. 235:318-325 (I960)), and Homo sapiens (Rous, Biochem. Biophys. Res. Commun. 69:74-78, (1976)), although the gene sequences encoding the corresponding enzymes are not known. Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have broad substrate specificity and thus represent suitable enzymes for hydrolyzing acetoacetyl-CoA. For example, the enzyme from Rattus norvegicus brain, acotl2 ( P 570103.1) (Robinson, Jr. et al., Biochem. Biophys. Res. Comm. 71 :959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. [00219] Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase which has been described to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3- hydroxyisobutyrate during valine degradation (Shimomura et al., J. Biol. Chem. 269: 14248- 14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura supra; Shimomura et al , 2000) and Homo sapiens (Shimomura et al., Methods Enzymol.

324:229-240 (2000)). Genes identified by sequence homology include hibch of Saccharomyces cerevisiae and BC 2292 of Bacillus cereus. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 19.

Table 19

[00220] Yet another hydrolase is the human dicarboxylic acid thioesterase, acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl- CoA (Westin et al, J. Biol. Chem. 280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which can also hydrolyze a broad range of CoA thiolesters ( aggert et al., J. Biol. Chem. 266: 11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana, Biochem. Int. 26:767-773 (1992)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 20.

Table 20

[00221] Other potential E. coli thiolester hydrolases include the gene products of tesA (Bonner et al., J. Biol. Chem. 247:3123-3133 (1972)), ^gC (Kuznetsova et al, FEMS Microbiol. Rev. 29:263-279 (2005); Zhuang et al, FEBS Lett. 516: 161-163 (2002)), paal (Song et al., J. Biol. Chem. 281 : 11028-1 1038 (2006)), andybdB (Leduc et al., J. Bacteriol. 189: 71 12-7126 (2007)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 21.

Table 21

[00222] In Figure 1 , step G, acetoacetate is decarboxylated to form acetone. This reaction can be catalyzed by acetoacetate decarboxylase (EC 4.1.1.4), an enzyme studied for its role in bacterial solventogenesis. Exemplary bacterial enzymes have been characterized from

Clostridium acetobutylicum (Benner et al., J. Am. Chem. So. 103:993-994 (1981); HIghbarger et al, Biochemistry 35:41-46 (1996); Petersen et al, Appl. Environ. Microbiol. 56:3491-3498 (1990); Rozzel et al. J. Am. Chem. 5Όα106:4937-4941 (1984)) and Clostridium beijerinckii (Ravagnani et al. Mol. Microbiol. 37: 1 172-1185 (2000)). Acetoacetate decarboxylase activity has also been demonstrated in Pseudomonas putida and Bacillus polymyxa but genes are not associated with this activity to date (Matiasek et al., Curr. Microbiol. 42: 276-281 (2001)). Bacterial genes in other organisms such as Clostridium botulinum and Bacillus

amyloliquefaciens FZB42 can be identified by sequence homology. In humans and other mammals, acetoacetate decarboxylase catalyzes the final step of the ketone -body pathway (Kalapos, Biochim. Biophys. Acta 1621 : 122-139 (2003)), but genes associated with this activity have not been identified to date. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 22.

Table 22

[00223] Other exemplary decarboxylase enzymes include pyruvate decarboxylase (EC 4.1.1.1) and benzoylformate decarboxylase (EC 4.1.1.7). Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3- hydroxypyruvate and 2-phenylpyruvate (Li et al., Biochemistry 38: 10004-10012 (1999)). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in i. coli (Killenberg-Jabs et al., Eur. J. Biochem. 268: 1698-1704 (2001); Li et al., supra; ter Schure et al., Appl. Environ. Micriobol. 64: 1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al., Protein Eng. Des. Sel, 18:345-357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs supra). Other well-characterized PDC enzymes include the enzymes from Acetobacter pasteurians (Chandra et ah, Arch. Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., Eur. J. Biochem.269:3256-3263 (2002)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 23.

Table 23

[00224] Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al, Biochemistry 42: 1820-1830 (2003); Hasson et al, Biochemistry 37:9918-9930 (1998)). Site- directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng. Des. Sel., 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Chembiochem 4:721-726 (2003); Lingen et al., Protein Eng. 15:585-593 (2002)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., FEMS Microbiol. Letters 34:57-60 (1986)). Additional genes from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl. Environ. Microbiol. 72:7510- 7517 (2006)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 24.

Table 24

[00225] A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., Proc. Natl. Acad. Sci. U.S.A.

102: 10670-10675 (2005)) has been cloned and has been functionally expressed in i. coli at Genomatica. KDC enzyme activity has been detected in several species of Rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka et al., Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the ^-terminus were sequenced

MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 13) (Shigeoka et al, supra). The gene can be identified by testing genes containing this ^-terminal sequence for KDC activity. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 25. Table 25

[00226] Hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (step B, Figure 1) is catalyzed by a crotonase (EC 4.2.1.55). These enzymes are part of the pathways for «-butanol formation in some organisms, particularly Clostridial species, and also comprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaea of the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genes encoding crotonase enzymes can be found in C. acetobutylicum (Boynton et al., Journal of Bacteriology 178:3015-3024 (1996)), C. kluyveri (Hillmer et al., FEBS Lett. 21 :351-354 (1972)), and Metallosphaera sedula (Berg et al., Archaea. Science. 318: 1782-1786 (2007)) though the sequence of the latter gene is not known. Enoyl-CoA hydratases, which are involved in fatty acid beta-oxidation and/or the metabolism of various amino acids, can also catalyze the hydration of crotonyl-CoA to form 3- hydroxybutyryl-CoA (Agnihotri et al., Med. Chem., 1 1 :9-20 (2003); Conrad et al., J Bacteriol. 118: 103-1 11 (1974)). The enoyl-CoA hydratases, phaA and phaB, of P. putida have been indicated to carry out the hydroxylation of double bonds during phenylacetate catabolism (Olivera et al, Proc. Natl. Acad. Sci USA 95:6419-6424 (1998)). The paaA and paaB from P. fluorescens catalyze analogous transformations (Olivera et al., supra). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park et al., J Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J Biochem. 270:3047-3054 (2003); Park et al, Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., supra; Park et al., supra; Park et al., supra). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 26.

Table 26

phaA ABF82233.1 Pseudomonas putida

phaB ABF82234.1 Pseudomonas putida

maoC NP 415905.1 Escherichia coli

paaF NP 415911.1 Escherichia coli

paaG NP 415912.1 Escherichia coli

[00227] Alternatively, the E. coli gene products of fadA and fadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Haller et al., Biochemistry, 39:4622-4629 (2000), Martinez-Carrion et al., J. Biol. Chem. 240:3538-3546 (1965) and Matties et al, Appl. Environ. Microbiol, 58: 1435-1439 (1992)). Knocking out a negative regulator encoded by fadR can be utilized to activate the fadB gene product (Jeng et al., Biochem. 13:2898-2903 (1974)). The fadl and fadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Atsumi et al., Nature 451 :86-89 (2008)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 27.

Table 27

[00228] The reversible conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA (step A, Figure 1) is catalyzed by the bifunctional enzyme 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ-isomerase. This enzyme first dehydrates 4-hydroxybutyryl-CoA to vinylacetyl-CoA, which subsequently rearranges to form crotonoyl-CoA. The enzymes from Clostridium kluyveri and C. aminobutyrium have been purified, characterized, and sequenced at the ^-terminal domain (Scherf et al, Eur. J. Biochem. 215:421-429 (1993); Scherf et al, Arch. Microbiol 161 :239-245 (1994)). The abfD genes from C. aminobutyrium and C. kluyveri match exactly with these n- terminal amino acid sequences, and have been indicated to encode the 4-hydroxybutyrul-CoA dehydratases/vinylacetyl-CoA Δ-isomerase activities. In addition, the abfD gene from

Porphyromonas gingivalis ATCC 33277 is identified through homology from genome projects. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 27.

Table 27

[00229] Dehydration of 3-hydroxyisobutyryl-CoA to methacrylyl-CoA (step R) can be accomplished by a reversible 3-hydroxyacyl-CoA dehydratase such as crotonase (also called 3- hydroxybutyryl-CoA dehydratase, EC 4.2.1.55) or enoyl-CoA hydratase (also called 3- hydroxyacyl-CoA dehydratase, EC 4.2.1.17). These enzymes are generally reversible

(Moskowitz et al, Biochemistry 8:2748-2755 (1969); Durre et al, FEMS Microbiol Rev 17:251- 262 (1995)). Exemplary enzymes are listed above. 3-hydroxyisobutyryl-CoA is not a natural substrate of these enzymes, but it is similar in structure to the native substrate, 3-hydroxybutyryl- CoA.

[00230] Referring to Figure 1, step M is carried out by isobutyryl-CoA mutase (ICM), a cobalamin-dependent methylmutase that reversibly rearranges the carbon backbone of butyryl- CoA into isobutyryl-CoA (RatnatiUeke et al, J Biol Chem 274:31679-31685 (1999)). Such an enzyme is also suitable for catalyzing the conversion of 4-hydroxybutyryl-CoA to 3- hydroxyisobutyryl-CoA, described by step Q of Figure 1. Genes encoding a heterodimeric ICM include icm and icmB of Streptomyces cinnamonensis (RatnatiUeke et al., supra; Vrijbloed et al., JBacteriol 181 :5600-5605 (1999); Zerbe-Burkhardt et al, J Biol Chem 273:6508-6517 (1998)). Homologous genes in Streptomyces avermitilis MA-4680 likely catalyze the same or similar transformations. A novel ICM-like enzyme in β-Proteobacterium LI 08 with nearly 100% identity with the corresponding peptide sequences from Methylibium petroleiphilum PM 1 was shown to convert 2-hydroxyisobutyryl-CoA to 3-hydroxybutyrate (Rohwerder et al., Appl Environ Microbiol 72:4128-4135 (2006)). This study also indicates that the replacement of the Phe residue in typical ICM enzymes (e.g., (RatnatiUeke et al., supra) with He at position 80 can be one of the mutations responsible for the activity on 2-hydroxyisobutyryl-CoA. This implies that if native ICM or ICM-like enzymes are unable to naturally catalyze the conversion of 4- hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA, they could be engineered to do so. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 28.

Table 28

[00231] Methylmalonyl-CoA mutase (MCM) enzymes represent an additional suitable class of enzymes for catalyzing the transformations described by steps P or T of Figure 3, provided they naturally exhibit or can be engineered to exhibit activity on butyryl-CoA or 4- hydroxybutyryl-CoA, respectively. MCM naturally catalyzes the conversion of succinyl-CoA into methylmalonyl-CoA. In E. coli, the reversible adenosylcobalamin-dependant mutase participates in a three-step pathway leading to the conversion of succinate to propionate (Haller et al., Biochemistry. 39:4622-4629 (2000)). MCM is encoded by genes scpA in Escherichia coli (Haller et al., supra; Bobik et al., Anal Bioanal Chem 375:344-349 (2003)) and mutA in Homo sapiens (Padovani et al., Biochemistry 45:9300-9306 (2006)). In several other organisms MCM contains alpha and beta subunits and is encoded by two genes. Exemplary genes encoding the two-subunit protein are Propionibacterium fredenreichii sp. shermani mutA and mutB and Methylobacterium extorquens mcmA and mcmB (Korotkova et al., J Biol Chem. 279: 13652- 13658 (2004)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 29. Table 29

[00232] These sequences can be used to identify homologue proteins in GenBank or other databases through sequence similarity searches (for example, BLASTp). The resulting homologue proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other suitable host microorganisms to generate production hosts. Additional genes include the following, which were identified based on high homology to the E. coli spcA gene product. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 30.

Table 30

[00233] There exists evidence that genes adjacent to the methylmalonyl-CoA mutase catalytic genes contribute to maximum activity. For example, it has been demonstrated that the meaB gene from M. extorquens forms a complex with methylmalonyl-CoA mutase, stimulates in vitro mutase activity, and possibly protects it from irreversible inactivation (Korotkova et al., supra). The M. extorquens meaB gene product is highly similar to the product of the E. coli argK gene (BLASTp: 45% identity, e-value: 4e-67), which is adjacent to scpA on the chromosome. No sequence for a meaB homolog in P. freudenreichii is catalogued in GenBank. However, the Propionibacterium acnes KPA171202 gene product, YP 055310.1, is 51% identical to the M. extorquens meaB protein and its gene is also adjacent to the methylmalonyl-CoA mutase gene on the chromosome. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 31.

Table 31

[00234] E. coli can synthesize adenosylcobalamin, a necessary cofactor for this reaction, when supplied with the intermediates cobinamide or cobalamin (Lawrence et al., J Bacteriol 177:6371- 6380 (1995); Lawrence et al, Genetics 142: 11-24 (1996)). Alternatively, the ability to synthesize cobalamins de novo has been conferred upon E. coli following the expression of heterologous genes (Raux et al., J Bacteriol 178:753-767 (1996)).

[00235] Step D in Figure 1 refers to an acid-thiol ligase which catalyzes the conversion of acetoacetyl-CoA to acetoacetate. An exemplary acid-thiol ligase is the enzyme encoded by sucCD of E. coli which 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)). Additional enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyze the ATP- dependant conversion of acetoacetate into acetoacetyl-CoA. Such enzymes can convert acetoacetyl-CoA to acetoacetate should they exhibit acetoacetyl-CoA hydrolase activity. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 32.

Table 32

[00236] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Although this enzyme has not been shown to react with acetoacetyl-CoA as a substrate, several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:27" '-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl- CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). The enzymes from fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra). The net transformation depicted by step D of Figure 1 can also be carried out by two enzymes such as acetate kinase and phosptransacetylase or butyrate kinase and phosphotransbutyrylase. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers shown in Table 33.

Table 33

[00237] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include a 4-hydroxybutyryl-CoA dehydratase encoded by one or more genes selected from the group consisting of fumA,fumB,fumC,fumH,fuml, MmcB, MmcC, hmd, BACCAP 02294, ANACOL 02527, NtherDRAFT 2368, dmdA, dmdB, crt, crtl, paaA, paaB, phaA,phaB, maoC, paaF, paaG, abfl), and Msed_1220. In particular embodiments the 4-hydroxybutyryl-CoA dehydratase is encoded by abfl).

[00238] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include a crotonase encoded by one or more genes selected from the group consisting of fumA,fumB,fumC,fumH,fuml, MmcB, MmcC, hmd,

BACCAP 02294, ANACOL 02527, NtherDRAFT 2368, dmdA, dmdB, crt, crt 1, paaA, paaB, phaA, phaB, maoC, paaF, paaG, abfl), and Msed_1220. In particular embodiments, the crotonase is encoded by one or more genes selected from the group consisting of crt, crt 1, paaA, paaB, phaA, phaB, maoC,paaF, and paaG.

[00239] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include a 3-hydroxybutyryl-CoA dehydrogenase encoded by one or more genes selected from the group consisting of thrA, akthr2, hom6, homl, hom2,fadB, fadJ, Hbd2, Hbdl, hbd, HSD17B10,phbB,phaB, Msed_1423, Msed 0399, Msed_0389, Msed 1993, adh, adhA, adh-A, mdh, IdhA, Idh, and bdh. In particular embodiments, the 3- hydroxybutyryl-CoA dehydrogenase is encoded by one or more genes selected from the group consisting of hbd, Hbd2, Hbdl, Msed_1423, Msed 0399, Msed_0389, Msed 1993, fadB, and fadJ.

[00240] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include an acetoacetyl-CoA synthetase encoded by one or more genes selected from the group consisting of sucC, sucD, AACS, AF1211, scs, and PAE3250. In particular embodiments, the acetoacetyl-CoA synthetase is encoded by one or more genes selected from the group consisting of sucC, sucD, AACS, and AF1211.

[00241] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include an acetyl-CoA:acetoacetate-CoA transferase encoded by one or more genes selected from the group consisting of atoA, atoD, act A, cg0592, ctfA, ctfB, catl, cat2, cat3, gctA, gctB, HPAG1 0676, HPAG1 0677, ScoA, and ScoB. In particular embodiments, the acetyl-CoA:acetoacetate-CoA transferase is encoded by one or more genes selected from the group consisting of atoA, atoD, act A, cg0592, ctfA, ctfB, HPAG1 0676, HPAG1 0677, ScoA, and ScoB. [00242] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include an acetoacetyl-CoA hydrolase encoded by one or more genes selected from the group consisting of acotl2, hibch, BC 2292, tesB, acot8, tesA, ybgC, paal, and ybdB. In particular embodiments, the acetoacetyl-CoA hydrolase is encoded by one or more genes selected from the group consisting of acotl2, hibch, and tesA.

[00243] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include an acetoacetate decarboxylase is encoded by one or more genes selected from the group consisting of pdc,pdcl, mdlC, dpgB, ilvB-l, kgd, kdcA, lysA, panD, dmpH, dmpE, xylll, xyllll, Reut B 5691, Reut B 5692, CAD, padl , pofK, (pad), padC, and pad. The non-naturally occurring microbial organism capable of producing isopropanol from 4- hydroxybutyryl-CoA can also include an acetoacetate decarboxylase encoded by one or more genes selected from the group consisting of Adc, cbei_3835, CLL A2135, RB AM 030030.

[00244] The non-naturally occurring microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA can include an acetone reductase encoded by one or more genes selected from the group consisting of thrA, akthr2, hom6, homl, hom2,fadB,fadJ, Hbd2, Hbdl, hbd, HSD17B10, phbB, phaB, Msed_1423, Msed 0399, Msed_0389, Msed 1993, adh, adhA, adh-A, mdh, IdhA, Idh, and bdh. In particular embodiments, the acetone reductase is encoded by one or more genes selected from the group consisting of adh, adhA, and adh-A.

[00245] The non-naturally occurring microbial organism capable of producing «-butanol from 4-hydroxybutyryl-CoA can include a 4-hydroxybutyryl-CoA dehydratase encoded by one or more genes selected from the group consisting of fumA,fumB,fumC,fumH,fuml, MmcB, MmcC, hmd, BACCAP 02294, ANACOL 02527, NtherDRAFT 2368, dmdA, dmdB, crt, crtl, paaA, paaB, phaA,phaB, maoC, paaF, paaG, abfl), and Msed_1220. In particular embodiments the 4-hydroxybutyryl-CoA dehydratase is encoded by abfl).

[00246] The non-naturally occurring microbial organism capable of producing «-butanol from 4-hydroxybutyryl-CoA can include a crotonoyl-CoA reductase encoded by one or more genes selected from the group consisting of bed, etfA, etfB, Ter, TDE0597, IBD, RPA3448, FadH, and enr. In particular embodiments, the crotonoyl-CoA reductase is encoded by one or more genes selected from the group consisting of bed, etfA, etfB, Ter, and TDE0597. [00247] The non-naturally occurring microbial organism capable of producing «-butanol from 4-hydroxybutyryl-CoA can include a butyryl-CoA reductase (aldehyde forming) encoded by one or more genes selected from the group consisting of acrl, sucD, bphG, adhE, Msed_0709, mcr, asd-2, Saci_2370, Aid, and eutE. In particular embodiments, the butyryl-CoA reductase

(aldehyde forming) is encoded by one or more genes selected from the group consisting of sucD, bphG, Msed_0709, mcr, and Aid.

[00248] The non-naturally occurring microbial organism capable of producing «-butanol from 4-hydroxybutyryl-CoA can include a butyraldehyde reductase encoded by one or more genes selected from the group consisting of air A, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhl, P84067, mmsb, dhat, and 3hidh. In particular embodiments, the butyraldehyde reductase is encoded by one or more genes selected from the group consisting of air A, ADH2, yqhD, bdh I, bdh II, 4hbd, adhl, and mmsb.

[00249] The non-naturally occurring microbial organism capable of producing «-butanol from 4-hydroxybutyryl-CoA can include a butyryl-CoA reductase (alcohol forming) encoded by one or more genes selected from the group consisting of adhE, adhE2, mcr, Rcas 2929,

NAP 1 02720, MGP2080 00535, and FAR. In particular embodiments, the butyryl-CoA reductase (alcohol forming) is encoded by one or more genes selected from the group consisting of adhE2, mcr, and FAR.

[00250] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a 4-hydroxybutyryl-CoA dehydratase encoded by one or more genes selected from the group consisting of fumA,fumB,fumC,fumH,fuml, MmcB, MmcC, hmd, BACCAP 02294, ANACOL 02527, NtherDRAFT 2368, dmdA, dmdB, crt, crtl, paaA, paaB, phaA,phaB, maoC, paaF, paaG, abfl), and Msed_1220. In particular

embodiments, the 4-hydroxybutyryl-CoA dehydratase is encoded by crt, crtl , paaA, paaB , phaA, phaB, maoC,paaF, and paaG.

[00251 ] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a crotonoyl-CoA reductase encoded by one or more genes selected from the group consisting of bed, etfA, etfB, Ter, TDE0597, IBD, RPA3448, FadH, and enr. In particular embodiments, the crotonoyl-CoA reductase is encoded by one or more genes selected from the group consisting of bed, etfA, etfB, Ter, and TDE0597.

[00252] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a isobutyryl-CoA mutase encoded by one or more genes selected from the group consisting of icm, icmB, icmA, Mpe BO 538, Mpe BO 541 , scpA, mutA, mutB, mcmA, mcmB, sbm, SARI 04585, YfreA 01000861, argK, PPA0597, and meaB. In particular embodiments, the isobutyryl-CoA mutase is encoded by one or more genes selected from the group consisting of icmB, icmA, Mpe B0538, and Mpe B0541.

[00253] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a 4-hydroxybutyryl-CoA mutase encoded by one or more genes selected from the group consisting of icm, icmB, icmA, Mpe B0538, Mpe B0541 , scpA, mutA, mutB, mcmA, mcmB, sbm, SARI 04585, YfreA 01000861, argK, PPA0597, and meaB. In particular embodiments the 4-hydroxybutyryl-CoA mutase is encoded by one or more genes selected from the group consisting of icmB, icmA, Mpe B0538, and Mpe B0541.

[00254] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a 3-hydroxyisobutyryl-CoA dehydratase encoded by one or more genes selected from the group consisting of fumA,fumB,fumC,fumH,fuml, MmcB, MmcC, hmd, BACCAP 02294, ANACOL 02527, NtherDRAFT 2368, dmdA, dmdB, crt, crtl, paaA, paaB, phaA,phaB, maoC, paaF, paaG, abfD, and Msed_1220. In particular

embodiments, the 3-hydroxyisobutyryl-CoA dehydratase is encoded by one or more genes selected from the group consisting of crt, crtl , paaA, paaB , phaA, phaB , maoC, paaF, and paaG.

[00255] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a methacrylyl-CoA reductase encoded by one or more genes selected from the group consisting of bed, etfA, etfB, Ter, TDE0597, IBD, RPA3448, FadH, and enr. In particular embodiments, the methacrylyl-CoA reductase is encoded by one or more genes selected from the group consisting of bed, etfA, etfB, Ter, and TDE0597.

[00256] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a isobutyryl-CoA reductase (aldehyde forming) encoded by one or more genes selected from the group consisting of acrl, sucD, bphG, adhE, Msed_0709, mcr, asd-2, Saci_2370, Aid, and eutE. In particular embodiments, the isobutyryl- CoA reductase (aldehyde forming) is encoded by one or more genes selected from the group consisting of sucD, bphG, Msed_0709, mcr, and Aid.

[00257] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include a isobutyraldehyde reductase encoded by one or more genes selected from the group consisting of alrA, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhi, P84067, mmsb, dhat, and 3hidh. In particular embodiments the isobutyraldehyde reductase is encoded by one or more genes selected from the group consisting of alrA, ADH2, yqhD, bdh I, bdh II, 4hbd, adhi, P 84067 , and mmsb.

[00258] The non-naturally occurring microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA can include an isobutyryl-CoA reductase (alcohol forming) encoded by one or more genes selected from the group consisting of adhE, adhE2, mcr, Rcas 2929, NAP 1 02720, MGP2080 00535, and FAR. In particular embodiments, the isobutyryl-CoA reductase (alcohol forming) is encoded by one or more genes selected from the group consisting of adhE2, mcr, and FAR.

[00259] Four requisite pathways to achieve the biosynthesis of 4-HB-CoA are exemplified herein and shown for purposes of illustration in Figure 2B. One requisite 4-HB-CoA

biosynthetic pathway includes the biosynthesis of 4-HB-CoA from succinate (the succinate pathway). The enzymes participating in this 4-HB-CoA pathway include CoA-independent succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase. In this pathway, CoA-independent succinic semialdehyde dehydrogenase catalyzes the reverse reaction. Another requisite 4-HB-CoA biosynthetic pathway includes the biosynthesis from succinate through succinyl-CoA (the succinyl-CoA pathway). The enzymes participating in this 4-HB-CoA pathway include succinyl-CoA synthetase, CoA-dependent succinic semialdehyde

dehydrogenase and 4-hydroxybutanoate dehydrogenase. Two other requisite 4-HB-CoA biosynthetic pathways include the biosynthesis of 4-HB-CoA from -ketoglutarate (the - ketoglutarate pathways). Hence, a third requisite 4-HB-CoA biosynthetic pathway is the biosynthesis of succinic semialdehyde through glutamate: succinic semialdehyde transaminase,

[00260] The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes participating in one or more 4-HB-CoA biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular 4-HB-CoA biosynthetic pathway can be expressed. For example, if a chosen host is deficient in both enzymes in the succinate to 4-HB-CoA pathway and this pathway is selected for 4-HB-CoA biosynthesis, then expressible nucleic acids for both CoA-independent succinic semialdehyde dehydrogenase and 4- hydroxybutanoate dehydrogenase are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous CoA-independent succinic semialdehyde dehydrogenase, but is deficient in 4-hydroxybutanoate dehydrogenase then an encoding nucleic acid is needed for this enzyme to achieve 4-HB-CoA biosynthesis.

[00261] 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, Anaerobiospirillum succiniciprodiicens, Actino bacillus succinogenes, Mannheimia succiniciprodiicens, 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 Upolytica, 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. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product.

[00262] In like fashion, where 4-HB-CoA biosynthesis is selected to occur through the succinate to succinyl-CoA pathway (the succinyl-CoA pathway), encoding nucleic acids for host deficiencies in the enzymes succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase and/or 4-hydroxybutanoate dehydrogenase are to be exogenously expressed in the recipient host. Selection of 4-HB-CoA biosynthesis through the -ketoglutarate to succinic semialdehyde pathway (the a-ketoglutarate pathway) can utilize exogenous expression for host deficiencies in one or more of the enzymes for glutamate: succinic semialdehyde transaminase, glutamate decarboxylase and/or 4-hydroxybutanoate dehydrogenase and 4-hydroxybutanoate dehydrogenase.

[00263] Depending on the 4-HB-CoA biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial 4-HB-CoA biocatalysts of the invention will include at least one exogenously expressed 4-HB-CoA pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more 4-HB-CoA biosynthetic pathways. For example, 4-HB-CoA biosynthesis can be established from all four pathways in a host deficient in 4-hydroxybutanoate dehydrogenase through exogenous expression of a 4-hydroxybutanoate dehydrogenase encoding nucleic acid. In contrast, 4-HB-CoA biosynthesis can be established from all four pathways in a host deficient in all seven enzymes through exogenous expression of all eight of CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, glutamate: succinic semialdehyde transaminase, glutamate decarboxylase, α-ketoglutarate decarboxylase and 4-hydroxybutanoate dehydrogenase.

[00264] 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 4-HB-CoA 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 or seven nucleic acids encoding the above enzymes constituting one or more 4-HB-CoA biosynthetic pathways. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize 4-HB-CoA 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 4-HB-CoA pathway precursors such as succinate, succinyl-CoA and/or -ketoglutarate.

[00265] In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize 4-HB. In this specific embodiment it can be useful to increase the synthesis or accumulation of a 4-HB-CoA pathway product to, for example, drive 4-HB-CoA pathway reactions toward 4-HB- CoAproduction. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described 4-HB-CoA pathway enzymes. Over expression of the 4-HB-CoA pathway enzyme or enzymes 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 4-HB-CoA producing microbial organisms of the invention through overexpression of one, two, three, four, five, six or all seven nucleic acids encoding 4-HB-CoAbiosynthetic pathway enzymes. 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 4-HB-CoAbiosynthetic pathway.

[00266] Non-naturally occurring microbial organisms also can be generated which biosynthesize isobutanol. As with the 4-HB-CoA producing microbial organisms of the invention, the isobutanol producing microbial organisms also can produce intracellularly or secret the isobutanol into the culture medium. Following the teachings and guidance provided previously for the construction of microbial organisms that synthesize 4-HB-CoA, additional isobutanol pathways can be incorporated into the 4-HB-CoA producing microbial organisms to generate organisms that also synthesize isobutanol and other isobutanol family compounds. The non-naturally occurring microbial organisms of the invention capable of isobutanol biosynthesis circumvent these chemical synthesis using 4-HB-CoA as an entry point as illustrated in Figure 2B. [00267] The additional isobutanol pathways to introduce into 4-HB-CoA producers include, for example, the exogenous expression in a host deficient background or the overexpression of one or more of the enzymes exemplified in Figure 2B. An initial step in the entry pathway to isobutanol is the conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA using 4- hydroxybutyrate:CoA transferase or the combination of butyrate kinase and

phosphotransbutyrylase. Accordingly, the additional initial isobutanol pathways to introduce into 4-HB-CoA producers to produce 4-hydroxybutyryl-CoA include, for example, the exogenous expression in a host deficient background or the overexpression of one or more of a 4-hydroxybutyrate: Co A transferase, butyrate kinase or phosphotransbutyrylase. In the absence of endogenous acyl-CoA synthetase capable of modifying 4-HB-CoA, the non-naturally occurring isobutanol producing microbial organisms can further include an exogenous acyl-CoA synthetase selective for 4-HB-CoA, or the combination of multiple enzymes that have as a net reaction conversion of 4-HB-CoAinto 4-HB-CoA. As exemplified further below in the

Examples, butyrate kinase and phosphotransbutyrylase exhibit isobutanol pathway activity and catalyze the conversions illustrated in Figure 2B with a 4-HB-CoAsubstrate. Therefore, these enzymes also can be referred to herein as 4-hydroxybutyrate kinase and

phosphotranshydroxybutyrylase respectively. Once 4-hydroxybutyryl-CoA is generated it can then be utilized for the biosynthesis of isobutanol following the subsequent steps shown Figure 2B.

Succinate - Succinyl-CoA (Succinyl-CoA synthetase or succinyl-CoA ligase)

[00268] Step A of Figure 2B involves CoA synthetase or ligase reactions with succinate as the substrate. Exemplary genes encoding enzymes likely to carry out these transformations include the sucCD genes of E. coli which naturally form a succinyl-CoA synthetase complex. This enzyme complex naturally catalyzes the formation of succinyl-CoA from succinate with the contaminant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). [00269] Additional exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical Journal 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395: 147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Bianco et al, J. Biol. Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate- CoA ligase from Bacillus subtilis (Boweret al, J. Bacteriol. 178(14):4122-4130 (1996)).

Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyze the ATP-dependant conversion of acetoacetate into acetoacetyl-CoA. 4-hydroxybutyryl-CoA synthetase activity has been demonstrated in Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)). This function has been tentatively assigned to the Msed 1422 gene.

[00270] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyrate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., 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 acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra).

Succinate - Succinyl-CoA (succinyl-CoA transferase)

[00271] Step A of Figure 2B can also be catalyzed by a transferase. The gene products of catl , cat2, and cat3 of Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4- hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activities (Seedorf et al. Proc Natl Acad Sci U.S.A. 105(6):2128-2133 (2008); Sohling and Gottschalk J Bacteriol 178(3):871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J.Biol.Chem. 283: 1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J.BioLChem. 279:45337-45346 (2004)).

[00272] An additionally useful enzyme for this type of transformation is acyl-CoA: acetate - CoA transferase, also known as acetate-CoA transferase (EC 2.8.3.8), which has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies and Schink Appl Environ Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al. Biochem.Biophys.Res Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel, supra (1968)). This enzyme is encoded by atoA (alpha subunit) and atoD (beta subunit) in E. coli sp. K12 (Korolev et al. Acta Crystallogr.D Biol Crystallogr. 58:21 16-2121 (2002); Vanderwinkel, supra (1968)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., 68:5186-5190 (2002)), Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)), and Clostridium acetobutylicum (Cary et al, 56: 1576-1583 (1990); Wiesenborn et al, 55:323-329 (1989)).

[00273] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack and Buckel FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl- CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al. Eur.J.Biochem.

118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mac et al.

Eur.J.Biochem. 226:41-51 (1994)).

[00274] Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate to succinyl- CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid. Exemplary succinyl-CoA:3:ketoacid- CoA transferases are present in Helicobacter pylori (Corthesy-Theulaz et al., J.Biol.Chem. 272:25659-25667 (1997)), Bacillus subtilis (Stols et al, Protein.Expr.Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics 68: 144-151 (2000); Tanaka et al., Mol.Hum.Reprod. 8: 16-23 (2002)). Information related to these proteins and genes is shown below:

Succinyl-CoA→ Succinic Semialdehyde (Succinyl-CoA reductase (aldehyde forming))

[00275] Step B of Figure 2 involves the conversion of succinyl-CoA to succinate

semialdehyde and is catalyzed by an aldehyde-forming succinyl-CoA reductase. Several acyl- CoA dehydrogenases are capable of reducing an acyl-CoA to its corresponding aldehyde.

Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase (Reiser and Somerville, J. Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M-l fatty acyl-CoA reductase (Ishige et al.

Appl.Environ.Microbiol. 68: 1192-1 195 (2002)), and a CoA- and NADP- dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri (Sohling and Gottschalk J Bacteriol 178:871-80 (1996); Sohling and Gottschalk J Bacteriol. 178:871-880 (1996)). SucD of P. gingivalis is another aldehyde-forming succinyl-CoA reductase (Takahashi et al. J.Bacteriol. 182:4704-4710 (2000)). The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski et al. J Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium

saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)). Gene Accession No. GI No. Organism

acrl YP 047869.1 50086359 Acinetobacter calcoaceticus

acrl AAC45217 1684886 Acinetobacter baylyi

acrl BAB85476.1 18857901 Acinetobacter sp. Strain M-l

sucD P38947.1 730847 Clostridium kluyveri

sucD NP 904963.1 34540484 Porphyromonas gingivalis

bphG BAA03892.1 425213 Pseudomonas sp

adhE AAV66076.1 55818563 Leuconostoc mes enter oides

bid AAP42563.1 31075383 Clostridium saccharoperbutylacetonicum

[00276] An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl- CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg et al. Science 318: 1782-1786 (2007); Thauer, R. K. Science 318: 1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al. J.Bacteriol. 188:8551-8559 (2006); Hugler et al. J.Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed 0709 in Metallosphaera sedula (Alber et al. J.Bacteriol. 188:8551-8559 (2006); Berg et al. Science 318: 1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al. J.Bacteriol. 188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate- semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent

dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including

Sulfolobus solfataricus and Sulfolobus acidocaldarius. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the aid gene from Clostridium beijerinckii (Toth et al., Appl

Environ.Microbiol 65:4973-4980 (1999)). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., Appl Environ.Microbiol 65:4973-4980 (1999)). mcr NP 378167.1 15922498 Sulfolobus tokodaii asd-2 NP 343563.1 15898958 Sulfolobus solfataricus

Saci 2370 YP 256941.1 70608071 Sulfolobus acidocaldarius

Aid AAT66436 49473535 Clostridium beijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P77445 2498347 Escherichia coli

Succinic Semialdehyde→ 4-hydroxybutyrate (4-hydroxybutyrate dehydrogenase)

[00277] Conversion of succinate semialdehyde to 4-hydroxybutyrate (Step C, Figure 2B) can be catalyzed by an oxidoreducatse that converts an aldehyde to alcohol. Exemplary genes encoding enzymes that catalyze the conversion of an aldehyde to alcohol, that is, alcohol dehydrogenase or equivalently aldehyde reductase, include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al. Appl.Environ.Microbiol. 66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al. Nature 451 :86-89 (2008)), yqhD from E. coli which has preference for molecules longer than C3 (Sulzenbacher et al. Journal of Molecular Biology 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyrylaldehyde into butanol (Walter et al. Journal of Bacteriology 174:7149-7158 (1992)). The protein sequences for each of these exemplary gene products, if available, can be found using the following GenBank accession numbers:

[00278] Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) have been characterized in Ralstonia eutropha (Bravo et al. J.Forensic Sci. 49:379-387 (2004), Clostridium kluyveri (Wolff et al. Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al. J.Biol.Chem. 278:41552-41556 (2003)). 4-HBd Q94B07 75249805 Arabidopsis thaliana

[00279] The adhl gene from Geobacillus thermoglucosidasius M10EXG (Jeon et al., J Biotechnol 135: 127-133 (2008)) has been indicated to exhibit high activity on both 4- hydroxybutanal and butanal. Thus this enzyme exhibits 1 ,4-butanediol dehydrogenase activity.

[00280] Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase which catalyzes the reversible oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde. This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al. J Mol Biol 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning et al. Biochem J 231 :481-484 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al. Methods Enzymol. 324:218- 228 (2000)) and Oryctolagus cuniculus (Chowdhury et al. Biosci.Biotechnol Biochem. 60:2043- 2047 (1996); Hawes et al. Methods Enzymol. 324:218-228 (2000)), mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhart et al. J Chem.Soc. [Perkin 1] 6: 1404- 1406 (1979); Chowdhury et al. Biosci.Biotechnol Biochem. 67:438-441 (2003); Chowdhury et al. Biosci.Biotechnol Biochem. 60:2043-2047 (1996)).

[00281] Several 3-hydroxyisobutyrate dehydrogenase enzymes have also been shown to convert malonic semialdehyde to 3-hydroxyproprionic acid (3-HP). Three gene candidates exhibiting this activity are mmsB from Pseudomonas aeruginosa PA01(62), mmsB from

Pseudomonas putida KT2440 (Liao et al., US Publication 2005/0221466) and mmsB from Pseudomonas putida E23 (Chowdhury et al., Biosci.Biotechnol.Biochem. 60:2043-2047 (1996)). An enzyme with 3-hydroxybutyrate dehydrogenase activity in Alcaligenes faecalis M3A has also been identified (Gokam et al., US Patent No. 7,393,676; Liao et al., US Publication No.

2005/0221466). Additional gene candidates from other organisms including Rhodobacter spaeroides can be inferred by sequence similarity.

[00282] The conversion of malonic semialdehyde to 3-HP can also be accomplished by two other enzymes: NADH-dependent 3-hydroxypropionate dehydrogenase and NADPH-dependent malonate semialdehyde reductase. An NADH-dependent 3-hydroxypropionate dehydrogenase is thought to participate in beta-alanine biosynthesis pathways from propionate in bacteria and plants (Rathinasabapathi, B. Journal of Plant Pathology 159:671-674 (2002); Stadtman, E. R. J.Am.Chem.Soc. 77:5765-5766 (1955)). This enzyme has not been associated with a gene in any organism to date. NADPH-dependent malonate semialdehyde reductase catalyzes the reverse reaction in autotrophic C02-fixing bacteria. Although the enzyme activity has been detected in Metallosphaera sedula, the identity of the gene is not known (Alber et al. J.Bacteriol. 188:8551- 8559 (2006)).

4-hydroxybutyrate - 4-hydroxybutyryl-phosphate (4-hydroxybutyrate kinase)

[00283] Exemplary kinases include the E. coli acetate kinase, encoded by ackA (Skarstedt and Silverstein J.Biol.Chem. 251 :6775-6783 (1976)), the C. acetobutylicum butyrate kinases, encoded by bukl and buk2 ((Walter et al. Gene 134(1): 107-1 11 (1993); Huang et al. J Mol Microbiol Biotechnol 2(l):33-38 (2000)), and the E. coli gamma-glutamyl kinase, encoded by proB (Smith et al. J.Bacteriol. 157:545-551 (1984)). These enzymes phosphorylate acetate, butyrate, and glutamate, respectively. The ackA gene product from E. coli also phosphorylates propionate (Hesslinger et al. Mol.Microbiol 27:477-492 (1998)). Information related to these proteins and genes is shown below:

4-hydroxybutyryl-CoA - 4-hydroxybutyryl-phosphate (Phosphotrans-4-hydroxybutyrylase)

[00284] Exemplary phosphate transferring acyltransferases that transform 4-hydroxybutyryl- CoA to 4-hydroxybutyryl-phosphate (step E, figure 2B) include phosphotransacetylase, encoded by pta, and phosphotransbutyrylase, encoded by ptb. The pta gene from E. coli encodes an enzyme that can convert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.

Biochim.Biophys.Acta 191 :559-569 (1969)). This enzyme can also utilize propionyl-CoA instead of acetyl-CoA forming propionate in the process (Hesslinger et al. Mol.Microbiol 27:477-492 (1998)). Similarly, the ptb gene from C. acetobutyhcum encodes an enzyme that can convert butyryl-CoA into butyryl-phosphate (Walter et al. Gene 134(1): p. 107-11 (1993)); Huang et al. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000). Additional ptb genes can be 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)).

Succinate→ Succinic Semialdehyde (Succinate reductase)

[00285] The conversion of succinate to succinate semialdehyde (Step F, figure 2B) can be catalyzed by a carboxylic acid reductase. 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. "Biocatalytic Reduction of Carboxylic Acids: Mechanism and Applications" Chapter 15 in Biocatalysis in the Pharmaceutical and Biotechnology Industries, ed. R.N. Patel, CRC Press LLC, Boca Raton, FL. (2006)).

[00286] Additional car and npt genes can be identified based on sequence homology.

[00287] 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, may be beneficial. Gene Accession No. GI No. Organism

griC 182438036 YP 001825755.1 Streptomyces griseus subsp. griseus NBRC

13350

griD 182438037 YP 001825756.1 Streptomyces griseus subsp. griseus NBRC

13350

[00288] 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 and no high-confidence hits were identified by sequence comparison homology searching.

Succinyl-CoA→ 4-hydroxybutyrate (Succinyl-CoA reductase (alcohol forming))

[00289] Step G in Figure 2B requires conversion of succinyl-CoA to 4-hydroxybutyrate. Exemplary two-step oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS. Lett., 281 :59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum

(Fontaine et al., J. BacterioL, 184:821-830 (2002)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol, 18:43-55 (1972); Koo et al, Biotechnol. Lett, 27:505-510 (2005)). Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers.

[00290] Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH- dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., J. BacterioL, 184:2404-2410 (2002); Strauss et al., Eur. J. Biochem., 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., supra). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms can have similar pathways (Klatt et al., Environ. Microbiol., 9:2067-2078 (2007)). Enzymes in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. Data related to the sequences for each of these exemplary gene products can be found using the following GenBank accession numbers.

[00291] Longer chain acyl-CoA molecules can be reduced by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et al, Plant Physiology, 122:635-644 (2000)) (FAR, AAD38039.1, 5020215, Simmondsia chinensis).

4-hydroxybutyrate→ 4-HB-CoA (4-hydroxybutyryl-CoA transferase)

[00292] 4-hydroxybutyryl CoA can be generated from 4-hydroxybutanoic using a 4- hydroxybutyryl-CoA transferase (step H, Figure 2B). Exemplary genes, organisms of origin, and reference citations are provided below. The gene products of catl, cat2, and cat3 of Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activities (Seedorf et al. Proc Natl Acad Sci U.S.A. 105(6):2128-2133 (2008); Sohling and Gottschalk J Bacteriol 178(3):871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., J.Biol.Chem. 283: 141 1-1418 (2008)) and Trypanosoma brucei (Riviere et al, J.Biol.Chem. 279:45337-45346 (2004)).

[00293] An additionally useful enzyme for this type of transformation is acyl-CoA: acetate - CoA transferase, also known as acetate-CoA transferase (EC 2.8.3.8), which has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies and Schink Appl Environ Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al. Biochem.Biophys.Res Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel, supra (1968)). This enzyme is encoded by atoA (alpha subunit) and atoD (beta subunit) in E. coli sp. K12 (Korolev et al. Acta Crystallogr.D Biol Crystallogr. 58:21 16-2121 (2002); Vanderwinkel, supra (1968)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., 68:5186-5190 (2002)), Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71 :58-68 (2007)), and Clostridium acetobutylicum (Cary et al, 56: 1576-1583 (1990); Wiesenborn et al, 55:323-329 (1989)).

[00294] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack and Buckel FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl- CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al. Eur.J.Biochem.

118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mac et al.

Eur.J.Biochem. 226:41-51 (1994)).

[00295] 4-hydroxybutyryl-CoA can be generated from 4-hydroxybutanoic using a synthetase or ligase enzyme (step H, Figure 2B). Exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochemical Journal 230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P.

chrysogenum (Lamas-Maceiras et al., Biochem. J. 395: 147-155 (2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Bianco et al, J. Biol. Chem. 265:7084-7090 (1990)), and the 6- carboxyhexanoate-CoA ligase from Bacillus subtilis (Boweret al., J. Bacteriol. 178(14):4122- 4130 (1996)). Additional candidate enzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyze the ATP- dependant conversion of acetoacetate into acetoacetyl-CoA. 4-hydroxybutyryl-CoA synthetase activity has been demonstrated in Metallosphaera sedula (Berg et al., Science 318: 1782-1786 (2007)). This function has been tentatively assigned to the Msed 1422 gene.

[00296] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is another candidate enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concurrent synthesis of ATP. Several enzymes with broad substrate specificities have been described in the literature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate, isobutyrate, isovalerate, succinate, fumarate, phenylacetate, indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., 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 acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Musfeldt et al., supra; Brasen et al., supra). Gene Accession No. GI No. Organism

AF1211 NP 070039.1 1 1498810 Archaeoglobus fiilgidiis DSM4304 scs YP 135572.1 55377722 Haloarcula marismortiii ATCC

43049

PAE3250 NP 560604.1 18313937 Pyrobaculiim aerophilum sir. IM2

[00297] Additional exemplary genes encoding enzymes likely to carry out these

transformations include the sucCD genes of E. coli which naturally form a succinyl-CoA synthetase complex. This enzyme complex naturally catalyzes the formation of succinyl-CoA from succinate with the contaminant consumption of one ATP, a reaction which is reversible in vivo (Buck et al, Biochem. 24:6245-6252 (1985)).

[00298] -KG→ Succinic Semialdehyde (step I, Figure 2B) (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase)

Glutamate dehydrogenase and 4-aminobutyrate dehydrogenase

[00299] Glutamate dehydrogenase and 4-aminobutyrate dehydrogenase can be catalyzed by aminating oxidoreductases. Enzymes in this EC class (1.4.1. a) catalyze the oxidative deamination of alpha-amino acids with NAD+ or NADP+ as acceptor, and the reactions are typically reversible. Exemplary oxidoreductases operating on amino acids include glutamate dehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh, and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhA gene product from Escherichia coli (Korber et al. J.Mol.Biol. 234: 1270-1273 (1993); McPherson and Wootton Nucleic.Acids Res. 11 :5257-5266 (1983)), gdh from Thermotoga maritima (Kort et al.

Extremophiles 1 :52-60 (1997); Lebbink, et al. J.Mol.Biol. 280:287-296 (1998)); Lebbink et al. J.Mol.Biol. 289:357-369 (1999)), and gdhAl from Halobacterium salinarum (Ingoldsby et al. Gene 349:237-244 (2005)) catalyze the reversible interconversion of glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both, respectively. The ldh gene of Bacillus cereus encodes the LeuDH protein that has a wide of range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula Biotechnol Bioeng. 68:557-562 (2000); Stoyan et al. J.Biotechnol 54:77-80 (1997)). The nadX gene from

Thermotoga maritime encoding for the aspartate dehydrogenase is involved in the biosynthesis of NAD (Yang et al. J.Biol.Chem. 278:8804-8808 (2003)).

[00300] Additional glutamate dehydrogenase gene candidates are found in Bacillus subtilis (Khan et al., Biosci.Biotechnol Biochem. 69: 1861-1870 (2005)), Nicotiana tabacum (Purnell et al, Planta 222: 167-180 (2005)), Oryza sativa (Abiko et al, Plant Cell Physiol 46: 1724-1734 (2005)), Haloferax mediterranei (Diaz et al., Extremophiles. 10: 105-115 (2006)) and

Halobactreium salinarum (Hayden et al., FEMS Microbiol Lett. 21 1 :37-41 (2002)). The Nicotiana tabacum enzyme is composed of alpha and beta subunits encoded by gdhl and gdh2 (Purnell et al., Planta 222: 167-180 (2005)). Overexpression of the NADH- dependent glutamate dehydrogenase was found to improve ethanol production in engineered strains of S. cerevisiae (Roca et al, Appl Environ.Microbiol 69:4732-4736 (2003)).

[00301] An exemplary enzyme for catalyzing the reversible conversion of aldehydes (e.g., succinate semialdehyde) to their corresponding primary amines is lysine 6-dehydrogenase (EC 1.4.1.18), encoded by the lysDH genes. The lysine 6-dehydrogenase (deaminating), encoded by lysDH gene, catalyze the oxidative deamination of the ε -amino group of L- lysine to form 2- aminoadipate-6-semialdehyde, which in turn nonenzymatically cyclizes to form ΔΙ-piperideine- 6-carboxylate (Misono and Nagasaki J.Bacteriol. 150:398-401 (1982)). The lysDH gene from Geobacillus stearothermophilus encodes a thermophilic NAD-dependent lysine 6-dehydrogenase (Heydari et al. Appl Environ.Microbiol 70:937-942 (2004)). The lysDH gene from Aeropyrum pernix Kl is identified through homology from genome projects. Additional enzymes can be found in Agrobacterium tumefaciens (Hashimoto et al., J Biochem. 106:76-80 (1989); Misono et al., J Bacteriol. 150:398-401 (1982)) and Achromobacter denitrificans (Ruldeekulthamrong et al, BMB.Rep. 41 :790-795 (2008)).

[00302] An enzyme that converts 3-oxoacids to 3-amino acids is 3,5-diaminohexanoate dehydrogenase (EC 1.4.1.1 1), an enzyme found in organisms that ferment lysine. The gene encoding this enzyme, kdd, was recently identified in Fusobacterium nucleatum (Kreimeyer et al., 282:7191-7197 (2007)). The enzyme has been purified and characterized in other organisms (Baker et al, 247:7724-7734 (1972); Baker et al, 13:292-299 (1974)) but the genes associated with these enzymes are not known. Candidates in Myxococcus xanthus, Porphyromonas gingivalis W83 and other sequenced organisms can be inferred by sequence homology.

Glutamate transaminase and 4-aminobutyrate transaminase

[00303] Aminotransferases reversibly convert an aldehyde or ketone to an amino group. Common amino donor/acceptor combinations include glutamate/alpha-ketoglutarate, alanine/pyruvate, and aspartate/oxaloacetate. Several enzymes have been shown to convert aldehydes to primary amines, and vice versa. Lysine-6-aminotransferase (EC 2.6.1.36) is one exemplary enzyme capable of forming a primary amine. This enzyme function, converting lysine to alpha-aminoadipate semialdehyde, has been demonstrated in yeast and bacteria. Candidates from Candida utilis (Hammer et al., J Basic Microbiol 32:21-27 (1992)), Flavobacterium lutescens (Fujii et al., J Biochem. 128:391-397 (2000)) and Streptomyces clavuligenus (Romero et al., J Ind.Microbiol Biotechnol 18:241-246 (1997)) have been characterized. A recombinant lysine-6-aminotransferase from S. clavuligenus was functionally expressed in E. coli (Tobin et al., J Bacteriol. 173:6223-6229 (1991)). The F. lutescens enzyme is specific to alpha- ketoglutarate as the amino acceptor (Soda et al., 7:4110-41 19 (1968)). Other enzymes which convert aldehydes to terminal amines include the dat gene product in Acinetobacter baumanii encoding 2,4-diaminobutanoate:2-ketoglutarate 4-transaminase (Ikai et al., J Bacteriol.

179:51 18-5125 (1997)). In addition to its natural substrate, 2,4-diaminobutyrate, DAT transaminates the terminal amines of lysine, 4-aminobutyrate and ornithine.

[00304] The conversion of an aldehyde to a terminal amine can also be catalyzed by gamma- aminobutyrate transaminase (GABA transaminase or 4-aminobutyrate transaminase). This enzyme naturally interconverts succinic semialdehyde and glutamate to 4-aminobutyrate and alpha-ketoglutarate and is known to have a broad substrate range (Schulz et al., 56: 1-6 (1990); Liu et al., 43: 10896-10905 (2004)). The two GABA transaminases in E. coli are encoded by gabT (Bartsch et al, J Bacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al, J.Biol.Chem. 280:4602-4608 (2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofa have been shown to react with a range of alternate substrates including 6- aminocaproic acid (Cooper, 113:80-82 (1985); SCOTT et al, 234:932-936 (1959)).

abat NP 999428.1 47523600 Sus scrofa

[00305] Additional enzyme candidates for interconverting aldehydes and primary amines are putrescine transminases or other diamine aminotransferases. The E. coli putrescine

aminotransferase is encoded by the ygjG gene and the purified enzyme also was able to transaminate cadaverine and spermidine (Samsonova et al., BMC.Microbiol 3:2 (2003)). In addition, activity of this enzyme on 1 ,7-diaminoheptane and with amino acceptors other than 2- oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been reported (Samsonova et al.,

BMC.Microbiol 3:2 (2003); KIM, 239:783-786 (1964)). A putrescine aminotransferase with higher activity with pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol. 184:3765-3773 (2002)).

[00306] Enzymes that transaminate 3-oxoacids include GABA aminotransferase (described above), beta-alanine/alpha-ketoglutarate aminotransferase and 3-amino-2-methylpropionate aminotransferase. Beta-alanine/alpha-ketoglutarate aminotransferase (WO08027742) reacts with beta-alanine to form malonic semialdehyde, a 3-oxoacid. The gene product of SkPYD4 in Saccharomyces kluyveri was shown to preferentially use beta-alanine as the amino group donor (Andersen et al., Gene 124: 105-109 (1993)). SkUGAl encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, UGAl (Ramos et al., Eur.J.Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in both beta-alanine and GABA transamination (Andersen and Hansen, Gene 124: 105-109 (1993)). 3-Amino-2-methylpropionate transaminase catalyzes the transformation from methylmalonate semialdehyde to 3-amino-2-methylpropionate. The enzyme has been characterized in Rattus norvegicus and Sus scrofa and is encoded by Abat (Tamaki et al, 324:376-389 (2000); Kakimoto et al, 156:374-380 (1968)).

Abat P50554.3 122065191 Rattus norvegicus

Abat P80147.2 120968 Sus scrofa

[00307] Several aminotransferases transaminate the amino groups of amino acids to form 2- oxoacids. Aspartate aminotransferase is an enzyme that naturally transfers an oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartate is similar in structure to OHED and 2-AHD. Aspartate aminotransferase activity is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al., 100:81-84 (1979); Yagi et al., 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al, 92:35-43 (1982)) and ASP5 from Arabidopsis thaliana (Kwok et al, 55:595-604 (2004); de la et al, 46:414-425 (2006); Wilkie et al, Protein Expr.Purif. 12:381-389 (1998)). The enzyme from Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., 19:4583-4589 (1980)). Aminotransferases that work on other amino-acid substrates may also be able to catalyze this transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes one such enzyme (Whalen et al., J.Bacteriol. 150:739- 746 (1982)). This gene product also catalyzes the transamination of alpha-ketobutyrate to generate a-aminobutyrate, although the amine donor in this reaction has not been identified (Whalen et al., J.Bacteriol. 158:571-574 (1984)). The gene product of the E. coli serC catalyzes two reactions, phosphoserine aminotransferase and phosphohydroxythreonme aminotransferase (Lam et al., J.Bacteriol. 172:6518-6528 (1990)), and activity on non-phosphorylated substrates could not be detected (Drewke et al, FEBS.Lett. 390: 179-182 (1996)).

[00308] Another enzyme candidate is alpha-aminoadipate aminotransferase (EC 2.6.1.39), an enzyme that participates in lysine biosynthesis and degradation in some organisms. This enzyme interconverts 2-aminoadipate and 2-oxoadipate, using alpha-ketoglutarate as the amino acceptor. Gene candidates are found in Homo sapiens (Okuno et al., Enzyme Protein 47: 136-148 (1993)) and Thermus thermophilus (Miyazaki et al., 150:2327-2334 (2004)). The Thermus thermophilus enzyme, encoded by lysN, is active with several alternate substrates including oxaloacetate, 2- oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate.

[00309] The decarboxylation of glutamate to 4-aminobutyrate is catalyzed by an amino acid decarboxylase such as glutamate decarboxylase (e.g., gadA, gadB, GADl). Another candidate is aspartate decarboxylase which participates in pantothenate biosynthesis and is encoded by gene panD in Escherichia coli (Dusch et al., Appl. Environ. Microbiol 65: 1530-1539 (1999); Merke and Nichols, FEMS Microbiol Lett. 143:247-252 (1996); Ramjee et al, Biochem. J 323 (Pt 3):661-669 (1997); and Schmitzberger et al, EMBO J 22:6193-6204 (2003)). Similar enzymes from Mycobacterium tuberculosis (Chopra et al., Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutamicum (Dusch et al., Appl. Environ. Microbiol 65: 1530-1539 (1999)) have been expressed and characterized in E. coli. Diaminopimelate decarboxylase (lysA), arginine decarboxylase (adiA, speA), ornithine decarboxylase (speF, speC) and lysine decarboxylase enzymes (e.g., cadA) are additional candidates to catalyze the decarboxylation of glutamate.

-KG→ Succinic Semialdehyde (alpha-ketoglutarate decarboxylase)

[00310] Alpha-ketoglutarate decarboxylase (Step I, Figure 2B) requires the decarboxylation of an alpha-ketoacid. The decarboxylation of keto-acids is catalyzed by a variety of enzymes with varied substrate specificities, including pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched- chain alpha- ketoacid decarboxylase.

[00311] Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (22). This enzyme has been extensively studied, engineered for altered activity, and functionally expressed in E. coli (Killenberg-Jabs et al, 268: 1698-1704 (2001); Li et al, Biochemistry. 38: 10004- 10012 (1999); ter Schure et al, Appl.Environ.Microbiol. 64: 1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc, also has a broad substrate range and has been a subject of directed engineering studies to alter the affinity for different substrates (Siegert et al., 18:345- 357 (2005)). The crystal structure of this enzyme is available (Killenberg-Jabs et al., 268: 1698- 1704 (2001)). Other well-characterized PDC candidates include the enzymes from Acetobacter pasteurians (Chandra et al., 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al., 269:3256-3263 (2002)).

[00312] Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al, 42: 1820-1830 (2003); Hasson et al, 37:9918-9930 (1998)). Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Ling en et al., Chembiochem. 4:721-726 (2003); Lingen et al, Protein Eng 15:585-593 (2002)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonas fluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl.Environ.Microbiol. 72:7510-7517 (2006)).

[00313] A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. The KDC from Mycobacterium tuberculosis (Tian et al., 102: 10670-10675 (2005)) has been cloned and functionally expressed in other internal projects at Genomatica. However, it is not an ideal candidate for strain engineering because it is large (-130 kDa) and GC-rich. KDC enzyme activity has been detected in several species of rhizobia including Bradyrhizobium japonicum and Mesorhizobium loti (Green et al, 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these organisms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka et al., 288:22-28 (1991)). The first twenty amino acids starting from the N- terminus were sequenced MTYKAPVKDVKFLLDKVFKV (Shigeoka and Nakano, 288:22-28 (1991)). The gene could be identified by testing candidate genes containing this N-terminal sequence for KDC activity. kgd NP 767092.1 27375563 Bradyrhizobium japoniciim kgd NP 105204.1 13473636 Mesorhizobium loti

[00314] A fourth candidate enzyme for catalyzing this reaction is branched chain alpha- ketoacid decarboxylase (BCKA). This class of enzyme has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku et al., 263: 18386-18396 (1988); Smit et al., 71 :303-311 (2005)). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2- oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smit et al., 71 :303-31 1 (2005)). The enzyme has been structurally characterized (Berg et al., 318: 1782-1786 (2007)). Sequence alignments between the Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; however, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, 263: 18386-18396 (1988)) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

[00315] Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the E 1 subunits of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al, 267: 16601-16606 (1992); Wynn et al, 267: 12400-12403 (1992); Wynn et al, 267: 1881- 1887 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al., 267: 12400- 12403 (1992)). These enzymes are composed of two alpha and two beta subunits. Gene Accession No. GI No. Organism

BCKDHB NP 898871.1 34101272 Homo sapiens

BCKDHA NP 000700.1 1 1386135 Homo sapiens

BCKDHB P21839 115502434 Bos taiirus

BCKDHA P11178 129030 Bos Taurus

[00316] 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 isopropanol, «-butanol, or isobutanol biosynthetic pathways.

Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol.

[00317] Depending on the isopropanol, «-butanol, or isobutanol 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 isopropanol, n- butanol, or isobutanol pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more isopropanol, «-butanol, or isobutanol biosynthetic pathways. For example, isopropanol, «-butanol, or isobutanol 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 an isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol can be included.

[00318] 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 isopropanol, «-butanol, or isobutanol 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, up to all nucleic acids encoding the enzymes or proteins constituting an isopropanol, «-butanol, or isobutanol biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize isopropanol, «-butanol, or isobutanol

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 isopropanol, «-butanol, or isobutanol pathway precursors such as acetyl-CoA.

[00319] Generally, a host microbial organism is selected such that it produces the precursor of an isopropanol, «-butanol, or isobutanol 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, acetyl-CoA 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 an isopropanol, «-butanol, or isobutanol pathway.

[00320] In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize isopropanol, n- butanol, or isobutanol. In this specific embodiment it can be useful to increase the synthesis or accumulation of an isopropanol, «-butanol, or isobutanol pathway product to, for example, drive isopropanol, «-butanol, or isobutanol pathway reactions toward isopropanol, «-butanol, or isobutanol production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described isopropanol, «-butanol, or isobutanol pathway enzymes or proteins. Over expression the enzyme or enzymes and/or protein or proteins of the isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol, through overexpression of one, two, three, four, five, that is, up to all nucleic acids encoding isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol biosynthetic pathway.

[00321] 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.

[00322] 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, isopropanol, «-butanol, or isobutanolbiosynthetic 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 isopropanol, «-butanol, or isobutanol biosynthetic capability. For example, a non- naturally occurring microbial organism having isopropanol, «-butanol, or isobutanol biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins. 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 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, 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.

[00323] In addition to the biosynthesis of isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol other than use of the isopropanol, n- butanol, or isobutanol producers is through addition of another microbial organism capable of converting isopropanol, «-butanol, or isobutanol pathway intermediate to isopropanol, «-butanol, or isobutanol. One such procedure includes, for example, the fermentation of a microbial organism that produces isopropanol, «-butanol, or isobutanol pathway intermediate. The isopropanol, «-butanol, or isobutanol pathway intermediate can then be used as a substrate for a second microbial organism that converts the isopropanol, «-butanol, or isobutanol pathway intermediate to isopropanol, «-butanol, or isobutanol. The isopropanol, «-butanol, or isobutanol pathway intermediate can be added directly to another culture of the second organism or the original culture of the isopropanol, «-butanol, or isobutanol 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.

[00324] 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, isopropanol, «-butanol, or isobutanol. 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 isopropanol, «-butanol, or isobutanol 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, isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol intermediate and the second microbial organism converts the intermediate to isopropanol, «-butanol, or isobutanol.

[00325] 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 isopropanol, «-butanol, or isobutanol.

[00326] Sources of encoding nucleic acids for isopropanol, «-butanol, or isobutanol 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, as well as other exemplary species disclosed herein 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 isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol 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.

[00327] In some instances, such as when an alternative isopropanol, «-butanol, or isobutanol biosynthetic pathway exists in an unrelated species, isopropanol, «-butanol, or isobutanol 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 can 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 isopropanol, «-butanol, or isobutanol.

[00328] 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, Anaerobiospirillum succiniciprodiicens, Actino bacillus succinogenes, Mannheimia succiniciprodiicens, 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 and Pichia pastoris. 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.

[00329] Methods for constructing and testing the expression levels of a non-naturally occurring isopropanol, «-butanol, or isobutanol-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).

[00330] Exogenous nucleic acid sequences involved in a pathway for production of isopropanol, «-butanol, or isobutanol 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. co\i or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an ^-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 i. 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.

[00331] An expression vector or vectors can be constructed to include one or more isopropanol, «-butanol, or isobutanol 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.

[00332] The invention provides a method for producing isopropanol, «-butanol, or isobutanol that includes culturing the non-naturally occurring microbial organism disclosed herein, under conditions and for a sufficient period of time to produce isopropanol, «-butanol, or isobutanol, including organisms that incorporate one, two, three, four, five, six, seven, eight, up to all exogenous nucleic acids encoding enzymes that complete a isopropanol, «-butanol, or isobutanol pathway. In some embodiments, at least one exogenous nucleic acid is a heterologous nucleic acid. Finally, culturing of thenon-naturally occurring microbial organisms of the invention can be performed in a substantially anaerobic culture medium. [00333] In some embodiments, a method for producing isobutanol includes culturing the non- naturally occurring microbial organisms disclosed herein under conditions and for a sufficient period of time to produce isobutanol. The method includes culturing a microbial organism having an isobutanol pathway that includes at least one exogenous nucleic acid encoding a isobutanol pathway enzyme expressed in a sufficient amount to produce isobutanol; the non- naturally occurring microbial organism further includes:

[00334] (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha- ketoglutarate: ferredoxin oxidoreductase;

[00335] (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate: ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an ¾ hydrogenase; or

[00336] (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an ¾ hydrogenase, and combinations thereof;

[00337] wherein when the non-naturally occurring microbial organism includes an isobutanol pathway that converts 4-hydroxybutryl-CoA to isobutanol, a 4-hydroxybutyryl pathway is selected from:

[00338] (I) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (aldehyde forming), a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[00339] (II) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (alcohol forming), a 4-Hydroxybutyrate kinase, a

Phosphotrans-4-hydroxybutyrylase; [00340] (III) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (aldehyde forming), a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[00341] (IV) a Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase), a Succinyl-CoA reductase (alcohol forming), a 4-Hydroxybutyryl-CoA transferase, or 4- Hydroxybutyryl-CoA synthetase;

[00342] (V) an Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase), a 4-Hydroxybutyrate dehydrogenase, a 4-Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[00343] (VI) an Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase; Glutamate decarboxylase; 4-aminobutyrate dehydrogenase and/or 4- aminobutyrate transaminase), a 4-Hydroxybutyrate dehydrogenase, a 4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[00344] (VII) a Succinate reductase, a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyrate kinase, a Phosphotrans-4-hydroxybutyrylase;

[00345] (VIII) a Succinate reductase, a 4-Hydroxybutyrate dehydrogenase, a 4- Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;

[00346] wherein the isobutanol pathway includes a pathway selected from:

[00347] (a) a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl- CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase;

[00348] (b) a 4-hydroxybutyryl-CoA dehydratase, a crotonoyl-CoA reductase, an isobutyryl- CoA mutase, and an isobutyryl-CoA reductase (alcohol forming); [00349] (c) a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, an isobutyryl-CoA reductase (aldehyde forming), and an isobutyraldehyde reductase;

[00350] (d) a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA-reductase, and an isobutyryl-CoA reductase (alcohol forming);

[00351] (e) an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA reductase (butyryl-CoA forming) an isobutyryl-CoA mutase, an isobutyryl-CoA reductase (aldehyde forming), and a branched-chain alcohol dehydrogenase;

[00352] (f) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase;

[00353] (g) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a valine dehydrogenase or transaminase, a valine decarboxylase, an omega transaminase, and a branched-chain alcohol dehydrogenase; and

[00354] (h) an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase, a branched-chain keto acid dehydrogenase, an isobutyryl-CoA reductase (aldehyde forming), and a branched-chain alcohol dehydrogenase.

[00355] The method that includes a microbial organism having pathway (i) can 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, ferredoxin, and combinations thereof.

[00356] The method that includes a microbial organism having pathway (ii) can further include 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, and combinations thereof. [00357] The method can include a microbial organism having two, three, four, five, six, or seven, eight, nine, or ten exogenous nucleic acids each encoding an isobutanol pathway enzyme.

[00358] The method that includes microbial organism having pathway (i) can include two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

[00359] The method that includes a microbial organism having pathway (ii) can include two, three or four exogenous nucleic acids each encoding a reductive TCA pathway enzyme.

[00360] The methods of the invention can include at least one exogenous nucleic acid that is a heterologous nucleic acid. The methods of the invention can include a non-naturally occurring microbial organism that is in a substantially anaerobic culture medium.

[00361] Suitable purification and/or assays to test for the production of isopropanol, n- butanol, or isobutanol 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 (see, for example, WO/2008/115840 and Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)).

[00362] The isopropanol, w-butanol, or isobutanol 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.

[00363] 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 isopropanol, «-butanol, or isobutanol producers can be cultured for the biosynthetic production of isopropanol, «-butanol, or isobutanol.

[00364] For the production of isopropanol, «-butanol, or isobutanol, 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 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 Patent application serial No. 1 1/891 ,602, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein.

[00365] 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.

[00366] In addition to renewable feedstocks such as those exemplified above, the isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source. [00367] 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 .

[00368] 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. H ₂ -dependent 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:

[00369] 2 C0 ₂ + 4 H ₂ + n ADP + n Pi→ CH ₃ COOH + 2 H ₂ 0 + n ATP

[00370] 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.

[00371] 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: methyltetrahydrofolate orrinoid 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 isopropanol, «-butanol, or isobutanol 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.

[00372] Additionally, 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 ¾ 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, 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 ¾ by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO 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 isopropanol, «-butanol, or isobutanol precursors, glyceraldehyde-3 -phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate: ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a isopropanol, «-butanol, or isobutanol 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 a reductive TCA pathway can confer syngas utilization ability.

[00373] 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, syngas, CO and/or C02. Such compounds include, for example, isopropanol, n- butanol, or isobutanol and any of the intermediate metabolites in the isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes isopropanol, «-butanol, or isobutanol when grown on a

carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the isopropanol, «-butanol, or isobutanol pathway when grown on a carbohydrate or other carbon source. The isopropanol, «-butanol, or isobutanol producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, acetyl-CoA.

[00374] 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 isopropanol, «-butanol, or isobutanol pathway enzyme or protein in sufficient amounts to produce isopropanol, «-butanol, or isobutanol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce isopropanol, «-butanol, or isobutanol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of isopropanol, «-butanol, or isobutanol resulting in intracellular concentrations between about 0.1- 200 mM or more. Generally, the intracellular concentration of isopropanol, «-butanol, or isobutanol 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. [00375] 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 isopropanol, «-butanol, or isobutanol producers can synthesize isopropanol, «-butanol, or isobutanol 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, isopropanol, «-butanol, or isobutanol producing microbial organisms can produce isopropanol, n- butanol, or isobutanol intracellularly and/or secrete the product into the culture medium.

[00376] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of isopropanol, «-butanol, or isobutanol 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 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM. [00377] 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 isopropanol, «-butanol, or isobutanol or any isopropanol, n- butanol, or isobutanol 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 isopropanol, n- butanol, or isobutanol or isopropanol, «-butanol, or isobutanol pathway intermediate including any isopropanol, «-butanol, or isobutanol impurities, or for side products generated in reactions diverging away from a isopropanol, «-butanol, or isobutanol pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens.

[00378] 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.

[00379] In some embodiments, a target isotopic ratio of an uptake source can be obtained via synthetic chemical enrichment of the uptake source. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory. In some embodiments, a target isotopic ratio of an uptake source can be obtained by choice of origin of the uptake source in nature. 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 C02, which can possess a larger amount of carbon- 14 than its petroleum-derived counterpart.

[00380] 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 ( ¹ 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".

[00381] 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.

[00382] 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 D6866-1 1 (effective April 1 , 201 1). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein. [00383] The biobased content of a compound is estimated by the ratio of carbon- 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 ¹ C/ ¹² C ratios of the blank, the sample and the modern reference, respectively. Fraction Modern is a measurement of the deviation of the ¹ 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 5 ¹³ C _VPDB =-19 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) ¹ C/ ¹² C ratio of 1.176 ± 0.010 x 10 ^~12 (Karlen et al., Arkiv Geojysik, 4:465-471 (1968)). The standard calculations take into account the differential uptake of one isotope with respect to another, for example, the preferential uptake in biological systems of C ¹² over C ¹³ over C ¹⁴ , and these corrections are reflected as a Fm corrected for δ .

[00384] 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 D6866-1 1 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. [00385] 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 D6866-1 1. 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.

[00386] 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% 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.

[00387] 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 biobased 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).

[00388] Accordingly, in some embodiments, the present invention provides isopropanol, n- butanol, or isobutanol or a isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol or a isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol or a isopropanol, «-butanol, or isobutanol intermediate that has a carbon- 12, carbon-13, and carbon- 14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the isopropanol, «-butanol, or isobutanol or a isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol or a isopropanol, «-butanol, or isobutanol 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.

[00389] Further, the present invention relates to the biologically produced isopropanol, n- butanol, or isobutanol or isopropanol, «-butanol, or isobutanol intermediate as disclosed herein, and to the products derived therefrom, wherein the isopropanol, «-butanol, or isobutanol or a isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol or a bioderived isopropanol, «-butanol, or isobutanol 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 isopropanol, «-butanol, or isobutanol or a bioderived isopropanol, «-butanol, or isobutanol intermediate as disclosed herein, wherein the bioderived product is chemically modified to generate a final product. Methods of chemically modifying a bioderived product of isopropanol, «-butanol, or isobutanol, 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 organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene -based products 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 organic solvents, polyurethane resins, polyester resins, hypoglycaemic agents, butadiene and/or butadiene -based products are generated directly from or in combination with bioderived isopropanol, «-butanol, or isobutanol or a bioderived isopropanol, «-butanol, or isobutanol intermediate as disclosed herein.

[00390] Isopropanol, «-butanol, or isobutanol are chemicals used in commercial and industrial applications and is also used as a raw material in the production of a wide range of products. Non-limiting examples of such applications and products include solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils, isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products. Accordingly, in some embodiments, the invention provides biobased used as a raw material in the production of a wide range of products comprising one or more bioderived isopropanol, «-butanol, or isobutanol or bioderived isopropanol, «-butanol, or isobutanol intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein. [00391] 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.

[00392] In some embodiments, the invention provides solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils, isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products comprising bioderived isopropanol, «-butanol, or isobutanol or bioderived isopropanol, «-butanol, or isobutanol intermediate, wherein the bioderived isopropanol, «-butanol, or isobutanol or bioderived isopropanol, «-butanol, or isobutanol intermediate includes all or part of the isopropanol, «-butanol, or isobutanol or isopropanol, n- butanol, or isobutanol intermediate used in the production of solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils, isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products. Thus, in some aspects, the invention provides biobased solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils,

isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products 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 isopropanol, n- butanol, or isobutanol or bioderived isopropanol, «-butanol, or isobutanol intermediate as disclosed herein. Additionally, in some aspects, the invention provides biobased solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils, isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products, wherein the isopropanol, «-butanol, or isobutanol or isopropanol, «-butanol, or isobutanol intermediate used in its production is a combination of bioderived and petroleum derived isopropanol, «-butanol, or isobutanol or isopropanol, «-butanol, or isobutanol intermediate. For example, biobased solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general-purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils, isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products can be produced using 50% bioderived isopropanol, «-butanol, or isobutanol and 50% petroleum derived isopropanol, «-butanol, or isobutanol 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 solvents, rubbing alcohol, , lacquers, thinners, inks, adhesives, general- purpose cleaners, disinfectants, cosmetics, toiletries, de-icers, pharmaceuticals, motor oils, isopropylamines, isopropylethers, isopropyl esters, butyl acrylate, butyl methacrylate, solution polymers, water— based latex coatings, enamels and lacquers, butyl acetate, glycol butyl esters, biofuels, isobutyl acetate, flavoring agents, plastics rubbers, paint, varnish removers and ink products using the bioderived isopropanol, «-butanol, or isobutanol or bioderived isopropanol, n- butanol, or isobutanol intermediate of the invention are well known in the art.

[00393] 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 an isopropanol, «-butanol, or isobutanol pathway enzyme or protein in sufficient amounts to produce isopropanol, «-butanol, or isobutanol. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce isopropanol, «-butanol, or isobutanol. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of isopropanol, «-butanol, or isobutanol resulting in intracellular concentrations between about 0.1- 2000 mM or more. Generally, the intracellular concentration of isopropanol, «-butanol, or isobutanol is between about 3-1800 mM, particularly between about 5-1700 mM and more particularly between about 8-1600 mM, including about 100 mM, 200 mM, 500 mM, 800 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.

[00394] 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. patent application No. US 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 conditions, the isopropanol, «-butanol, or isobutanol producers can synthesize isopropanol, «-butanol, or isobutanol 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, isopropanol, «-butanol, or isobutanol producing microbial organisms can produce isopropanol, «-butanol, or isobutanol intracellularly and/or secrete the product into the culture medium.

[00395] In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of isobutanol and other products disclosed herein 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.

[00396] 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.

[00397] As described herein, one exemplary growth condition for achieving biosynthesis of isopropanol, «-butanol, or isobutanol 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 refer 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.

[00398] The culture conditions described herein can be scaled up and grown continuously for manufacturing of isopropanol, «-butanol, or isobutanol. 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 isopropanol, «-butanol, or isobutanol. Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of isopropanol, «-butanol, or isobutanol will include culturing a non-naturally occurring isopropanol, «-butanol, or isobutanol 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.

[00399] Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of isopropanol, «-butanol, or isobutanol 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.

[00400] In addition to the above fermentation procedures using the isopropanol, «-butanol, or isobutanol producers of the invention for continuous production of substantial quantities of isopropanol, «-butanol, or isobutanol, the isopropanol, «-butanol, or isobutanol 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.

[00401] 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

[00402] In some embodiments, syngas can be used as a carbon feedstock. Important process considerations for a syngas fermentation are high biomass concentration and good gas-liquid mass transfer (Bredwell et al., Biotechnol Prog. 15:834-844 (1999). The solubility of CO in water is somewhat less than that of oxygen. Continuously gas-sparged fermentations can be performed in controlled fermenters with constant off-gas analysis by mass spectrometry and periodic liquid sampling and analysis by GC and HPLC. The liquid phase can function in batch mode. Fermentation products such as alcohols, organic acids, and residual glucose along with residual methanol are quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm). All piping in these systems is glass or metal to maintain anaerobic conditions. The gas sparging is performed with glass frits to decrease bubble size and improve mass transfer. Various sparging rates are tested, ranging from about 0.1 to 1 vvm (vapor volumes per minute). To obtain accurate measurements of gas uptake rates, periodic challenges are performed in which the gas flow is temporarily stopped, and the gas phase composition is monitored as a function of time.

[00403] In order to achieve the overall target productivity, methods of cell retention or recycle are employed. One method to increase the microbial concentration is to recycle cells via a tangential flow membrane from a sidestream. Repeated batch culture can also be used, as previously described for production of acetate by Moorella (Sakai et al., J Biosci.Bioeng 99:252- 258 (2005)). Various other methods can also be used (Bredwell et al., Biotechnol Prog. 15:834- 844 (1999); Datar et al, Biotechnol Bioeng 86:587-594 (2004)). Additional optimization can be tested such as overpressure at 1.5 atm to improve mass transfer (Najafpour and Younesi, Enzyme and Microbial Technology 38[l-2], 223-228 (2006)). [00404] Once satisfactory performance is achieved using pure H ₂ /CO as the feed, synthetic gas mixtures are generated containing inhibitors likely to be present in commercial syngas. For example, a typical impurity profile is 4.5% CH ₄ , 0.1% C ₂ H ₂ , 0.35% C ₂ H ₆ , 1.4% C ₂ H ₄ , and 150 ppm nitric oxide (Datar et al., Biotechnol Bioeng 86:587-594 (2004)). Tars, represented by compounds such as benzene, toluene, ethylbenzene, p-xylene, o-xylene, and naphthalene, are added at ppm levels to test for any effect on production. For example, it has been shown that 40 ppm NO is inhibitory to C. carboxidivorans (Ahmed and Lewis, Biotechnol Bioeng 97: 1080- 1086 (2007)). Cultures are tested in shake-flask cultures before moving to a fermentor. Also, different levels of these potential inhibitory compounds are tested to quantify the effect they have on cell growth. This knowledge is used to develop specifications for syngas purity, which is utilized for scale up studies and production. If any particular component is found to be difficult to decrease or remove from syngas used for scale up, an adaptive evolution procedure is utilized to adapt cells to tolerate one or more impurities.

[00405] Advances in the field of protein engineering make it feasible to alter any of the enzymes disclosed herein to act efficiently on substrates not known to be natural to them. Below are several examples of broad-specificity enzymes from diverse classes of interest and methods that have been used for evolving such enzymes to act on non-natural substrates.

[00406] One class of enzymes in the pathways disclosed herein is the oxidoreductases that interconvert ketones or aldehydes to alcohols (1.1.1). Enzymes in this class that can operate on a wide range of substrates. An alcohol dehydrogenase (1.1.1.1) purified from the soil bacterium Brevibacterium sp KU 1309 (Hirano et al., J. Biosci. Bioeng. 100:318-322 (2005)) was shown to operate on a plethora of aliphatic as well as aromatic alcohols with high activities. Table 34 shows the activity of the enzyme and its K _m on different alcohols. The enzyme is reversible and has very high activity on several aldehydes also as shown in Table 34.

Table 34

(i?)-2-Phenylpropanol 63 0.020

Benzyl alcohol 199 0.012

3 -Pheny lpr opanol 135 0.033

Ethanol 76

1 -Butanol 111

1-Octanol 101

1-Dodecanol 68

1 -Phenylethanol 46

2-Propanol 54

In this Table, the activity of 2-phenylethanol, corresponding to 19.2 U/mg, was taken

Table 35

[00408] Lactate dehydrogenase (1.1.1.27) from Ralstonia eutropha is another enzyme that has been demonstrated to have high activities on several 2-oxoacids such as 2-oxobutyrate, 2- oxopentanoate and 2-oxoglutarate (a C5 compound analogous to 2-oxoadipate) (Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). Column 2 in Table 36 shows the activities of IdhA from ?. eutropha (formerly .4. eutrophus) on different substrates (Steinbuchel et al., supra).

Table 36

Substrate Activity of

L(+ L(+ D(- lactate dehydro-genase lactate dehydrolactate dehydro-genase from eustrophiis genase from rabbit from Z. leichmanii muscle

2-Oxobutyrate 107.0 18.6 1.1

2-Oxovalerate 125.0 0.7 0.0

3-Methyl-2- 28.5 0.0 0.0 oxobutyrate

3-Methyl-2- 5.3 0.0 0.0 oxovalerate

4-Methyl-2- 39.0 1.4 1.1 oxopentanoate

Oxaloacetate 0.0 33.1 23.1

2-Oxoglutarate 79.6 0.0 0.0

3 -Fluoropyruvate 33.6 74.3 40.0

[00409] Oxidoreductases that can convert 2-oxoacids to their acyl-CoA counterparts (1.2.1) have been shown to accept multiple substrates as well. For example, branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as 2-oxoisovalerate dehydrogenase (1.2.1.25), participates in branched-chain amino acid degradation pathways, converting 2-keto acids derivatives of valine, leucine and isoleucine to their acyl-CoA derivatives and C0 ₂ . In some organisms including Rattus norvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al., Biochem. Mol. Biol. Int. 31 :911-922 (1993)), this complex has been shown to have a broad substrate range that includes linear oxo-acids such as 2- oxobutanoate and alpha-ketoglutarate, in addition to the branched-chain amino acid precursors.

[00410] CoA transferases (2.8.3) have been demonstrated to have the ability to act on more than one substrate. Specifically, a CoA transferase was purified from Clostridium acetobutylicum and was reported to have the highest activities on acetate, propionate, and butyrate. It also had significant activities with valerate, isobutyrate, and crotonate (Wiesenborn et al., Appl. Environ. Microbiol. 55:323-329 (1989)). In another study, the E. coli enzyme acyl-CoA:acetate-CoA transferase, also known as acetate-CoA transferase (EC 2.8.3.8), has been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., Appl Environ Microbiol 58: 1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., supra).

[0041 1] The argument for broad substrate specificity of the enzymes is further strengthened as we explore other enzyme classes. Some isomerases (5.3.3) have also been proven to operate on multiple substrates. For example, L-rhamnose isomerase from Pseudomonas stutzeri catalyzes the isomerization between various aldoalses and ketoses (Yoshida et al., J. Mol. Biol. 365: 1505- 1516 (2007)). These include isomerization between L-rhamnose and L-rhamnulose, L-mannose and L-fructose, L-xylose and L-xylulose, D-ribose and D-ribulose, and D-allose and D-psicose.

[00412] In yet another class of enzymes, the phosphotransferases (2.7.1), the homoserine kinase (2.7.1.39) from i. coli that converts L-homoserine to L-homoserine phosphate, was found to phosphorylate numerous homoserine analogs. In these substrates, the carboxyl functional group at the R-position had been replaced by an ester or by a hydroxymethyl group (Huo et al., Biochemistry 35: 16180-16185 (1996)). Table 37 demonstrates the broad substrate specificity of this kinase.

Table 37

^-propyl ester

L-homoserine 16.4 ± 0.8 84 6.9 ± 1.1 2.4 ± 0.3 isobutyl ester

L-homoserine n- 29.1 ± 1.2 160 5.8 ± 0.8 5.0 ± 0.5 butyl ester

[00413] Another class of enzymes t lat we have encountered in the proposec pathways is the acid-thiol ligases (6.2.1). Like enzymes in other classes, certain enzymes in this class have been determined to have broad substrate specificity. For example, acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez- Valverde et al., Appl. Environ. Microbiol. 59: 1149-1 154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium trifolii could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., J. Am. Chem. Soc. 123:5822-5823 (2001)). In a similar vein, we also found decarboxylases (4.1.1) with broad substrate ranges. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme isolated from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2- ketovalerate, and 2-phenylpyruvate (Li et al., Biochemistry 38: 10004-10012 (1999)). Similarly, benzoylformate decarboxylase has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al., Biochemistry 42: 1820-1830 (2003);Hasson et al, Biochemistry 37:9918-9930 (1998)). Branched chain alpha-ketoacid decarboxylase (BCKA) has been shown to act on a variety of compounds varying in chain length from 3 to 6 carbons (Oku et al, J Biol Chem. 263: 18386-18396 (1988)). The enzyme in

Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 4- methyl-2-oxobutanoate and isocaproate (Smit et al., Appl Environ Microbiol 71 :303-311 (2005)).

[00414] Interestingly, enzymes known to have one dominant activity have also been indicated to catalyze a very different function. For example, the cofactor-dependent phosphoglycerate mutase (5.4.2.1) from Bacillus stearothermophilus and Bacillus subtilis is known to function as a phosphatase as well (Rigden et al., Protein Sci. 10: 1835-1846 (2001)). The enzyme from ?. stearothermophilus is known to have activity on several substrates, including 3- phosphoglycerate, alpha-napthylphosphate, p-nitrophenylphosphate, AMP, fructose-6-phosphate, ribose-5-phosphate and CMP.

[00415] In contrast to these examples, where the enzymes naturally have broad substrate specificities, numerous enzymes have been modified using directed evolution to broaden their specificity towards their non-natural substrates. Alternatively, the substrate preference of an enzyme has also been changed using directed evolution. The success of these methods has been well-documented in literature and reinforces our claim that it is more than feasible to engineer a given enzyme for efficient function on a natural/non-natural substrate. For example, it has been reported that the enantioselectivity of a lipase from Pseudomonas aeruginosa was improved significantly. This enzyme hydrolyzed p-nitrophenyl 2-methyldecanoate with only 2%

enantiomeric excess (ee) in favor of the (S)-aci ^' d. However, after four successive rounds of error- prone mutagenesis and screening, a variant was produced that catalyzed the requisite reaction with 81% ee (Reetz et al, Angew. Chem. Int. Ed Engl. 36:2830-2832 (1997)).

[00416] As mentioned earlier, directed evolution methods have enabled the modification of an enzyme to function on an array of substrates not natural to it. The substrate specificity of the lipase in P. aeruginosa was broadened by randomization of amino acid residues near the active site. This allowed for the acceptance of alpha-substituted carboxylic acid esters by this enzyme (Reetz et al., Angew. Chem. Int. Ed Engl. 44:4192-4196 (2005)). In another successful attempt, DNA shuffling was employed to create an Escherichia coli aminotransferase that accepted β- branched substrates, which were poorly accepted by the wild-type enzyme (Yano et al., Proc. Natl. Acad. Sci. U. S. A 95:551 1-5515 (1998)). Specifically, at the end of four rounds of shuffling, the activity of aspartate aminotransferase for valine and 2-oxovaline increased by up to five orders of magnitude, while decreasing the activity towards the natural substrate, aspartate, by up to 30-fold. In a very recent report in Science, an algorithm was used to design a retro- aldolase that could be used to catalyze the carbon-carbon bond cleavage in a non-natural and non-biological substrate, 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone. These algorithms used different combinations of four different catalytic motifs to design new enzymes and 20 of the selected designs for experimental characterization had four-fold improved rates over the uncatalyzed reaction (Jiang et al., Science 319: 1387-1391 (2008)). Thus, not only are these engineering approaches capable of expanding the array of substrates on which an enzyme can act, but they enable the design and construction of very efficient enzymes. For example, a method of DNA shuffling (random chimeragenesis on transient templates or RACHITT) was reported to lead to an engineered monooxygenase that had an improved rate of desulfurization on complex substrates as well as 20-fold faster conversion of a non-natural substrate (Coco et al. Nat. Biotechnol. 19:354-359 (2001)). Similarly, the specific activity of a sluggish mutant triosephosphate isomerase enzyme was improved up to 19-fold from 1.3 fold (Hermes et al., Proc. Natl. Acad. Sci. U. S. A 87:696-700 (1990)). This enhancement in specific activity was accomplished by using random mutagenesis over the whole length of the protein and the improvement could be traced back to mutations in six amino acid residues.

[00417] The effectiveness of protein engineering approaches to alter the substrate specificity of an enzyme for a desired substrate has also been demonstrated in several studies.

Isopropylmalate dehydrogenase from Thermus thermophilus was modified by changing residues close to the active site so that it could now act on malate and D-lactate as substrates (Fujita et al., Biosci. Biotechnol Biochem. 65:2695-2700 (2001)). In this study as well as in others, it was pointed out that one or a few residues could be modified to alter the substrate specificity. A case in point is the dihydroflavonol 4-reductase for which a single amino acid was changed in the presumed substrate-binding region that could preferentially reduce dihydrokaempferol (Johnson et al., Plant J. 25:325-333 (2001)). The substrate specificity of a very specific isocitrate dehydrogenase from Escherichia coli was changed from isocitrate to isopropylmalate by changing one residue in the active site (Doyle et al., Biochemistry 40:4234-4241 (2001)). In a similar vein, the cofactor specificity of a NAD ⁺ -dependent 1 ,5-hydroxyprostaglandin

dehydrogenase was altered to NADP ⁺ by changing a few residues near the N-terminal end (Cho et al., Arch. Biochem. Biophys. 419: 139-146 (2003)). Sequence analysis and molecular modeling analysis were used to identify the key residues for modification, which were further studied by site-directed mutagenesis.

[00418] There are many other examples spanning diverse classes of enzymes where the function of enzyme was changed to favor one non-natural substrate over the natural substrate of the enzyme. A fucosidase was evolved from a galactosidase in E. coli by DNA shuffling and screening (Zhang et al, Proc Natl Acad Sci U S. A 94:4504-4509 (1997)). Similarly, aspartate aminotransferase from E. coli was converted into a tyrosine aminotransferase using homology modeling and site-directed mutagenesis (Onuffer et al., Protein Sci. 4: 1750-1757 (1995)). Site- directed mutagenesis of two residues in the active site of benzoylformate decarboxylase from P. putida reportedly altered the affinity (K _m ) towards natural and non-natural substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). Cytochrome c peroxidase (CCP) from

Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and up to 1000-fold increased specificity for this substrate relative to that for the natural substrate (Iffland et al., Biochemistry 39: 10790-10798 (2000)).

[00419] In some cases, enzymes with different substrate preferences than both the parent enzymes have been obtained. For example, biphenyl-dioxygenase-mediated degradation of polychlorinated biphenyls was improved by shuffling genes from two bacteria, Pseudomonas pseudoalcaligens and Burkholderia cepacia (Kumamaru et al., Nat. Biotechnol 16, 663-666 (1998)). The resulting chimeric biphenyl oxygenases showed different substrate preferences than both the parental enzymes and enhanced the degradation activity towards related biphenyl compounds and single aromatic ring hydrocarbons such as toluene and benzene which were originally poor substrates for the enzyme.

[00420] It is not only possible to change the enzyme specificity, but also to enhance the activities on those substrates on which the enzymes naturally have low activities. One study demonstrated that amino acid racemase from P. putida that had broad substrate specificity (on lysine, arginine, alanine, serine, methionine, cysteine, leucine and histidine among others) but low activity towards tryptophan could be improved significantly by random mutagenesis ( Kino et al, Appl. Microbiol. Biotechnol. 73: 1299-1305 (2007)). Similarly, the active site of the bovine BCKAD was engineered to favor alternate substrate acetyl-CoA ( Meng et al.

Biochemistry 33: 12879-12885 (1994)). An interesting aspect of these approaches is that even if random methods have been applied to generate these mutated enzymes with efficacious activities, the exact mutations or structural changes that confer the improvement in activity can be identified. For example, in the aforementioned study, the mutations that facilitated improved activity on tryptophan could be traced back to two different positions.

[00421] Directed evolution has also been used to express proteins that are difficult to express. For example, by subjecting the horseradish peroxidase to random mutagenesis and gene recombination, mutants could be extracted that had more than 14-fold activity than the wild type (Lin et al, Biotechnol. Prog. 15:467-471 (1999)).

[00422] A final example of directed evolution shows the extensive modifications to which an enzyme can be subjected to achieve a range of desired functions. The enzyme, lactate dehydrogenase from Bacillus stearothermophilus was subjected to site-directed mutagenesis, and three amino acid substitutions were made at sites that were believed to determine the specificity towards different hydroxyacids (Clarke et al., Biochem. Biophys. Res. Commun. 148: 15-23 (1987)). After these mutations, the specificity for oxaloacetate over pyruvate was increased to 500 in contrast to the wild type enzyme that had a catalytic specificity for pyruvate over oxaloacetate of 1000. This enzyme was further engineered using site-directed mutagenesis to have activity towards branched-chain substituted pyruvates (Wilks et al., Biochemistry 29:8587- 8591 (1990)). Specifically, the enzyme had a 55-fold improvement in Kc _at for alpha- ketoisocaproate. Three structural modifications were made in the same enzyme to change its substrate specificity from lactate to malate. The enzyme was highly active and specific towards malate (Wilks et al., Science 242: 1541-1544 (1988)). The same enzyme from ?.

stearothermophilus was subsequently engineered to have high catalytic activity towards alpha- keto acids with positively charged side chains, such as those containing ammonium groups (Hogan et al., Biochemistry 34:4225-4230 (1995)). Mutants with acidic amino acids introduced at position 102 of the enzyme favored binding of such side chain ammonium groups. The results obtained proved that the mutants showed up to 25-fold improvements in kc _a t/ _m values for omega-amino-alpha-keto acid substrates. This enzyme was also structurally modified to function as a phenyllactate dehydrogenase instead of a lactate dehydrogenase (Wilks et al., Biochemistry 31 :7802-7806 (1992)). Restriction sites were introduced into the gene for the enzyme which enabled a region of the gene to be excised. This region coded for a mobile surface loop of polypeptide (residues 98-110) which normally seals the active site vacuole from bulk solvent and is a major determinant of substrate specificity. The variable length and sequence loops were inserted into the cut gene and used to synthesize hydroxyacid dehydrogenases with altered substrate specificities. With one longer loop construction, activity with pyruvate was reduced one-million-fold but activity with phenylpyruvate was largely unaltered. A switch in specificity (kcat/Km) of 390,000-fold was achieved. The 1700: 1 selectivity of this enzyme for

phenylpyruvate over pyruvate is that required in a phenyllactate dehydrogenase.

[00423] As indicated above, 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 (e.g., >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.

[00424] Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol.Eng 22: 1 1-19 (2005); Huisman et al., Enzyme evolution for chemical process applications, p. 717-742 (2007)). In R. N. Patel (ed.), Biocatalysis in the pharmaceutical and biotechnology industries. CRC Press; Otten, et al., 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.

[00425] 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 ) - broadens substrate binding to include non-natural substrates; inhibition (¾) - to remove inhibition by products, substrates, or key intermediates; activity (kcat) - increases enzymatic reaction rates to achieve desired flux; expression levels - increases 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.

[00426] The following exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Any of these can be used to alter/optimize activity of a decarboxylase enzyme.

[00427] EpPCR (Pritchard et al, J Theor.Biol 234:497-509 (2005)) introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions by the addition of Mn ²⁺ ions, by biasing dNTP concentrations, or by other conditional variations. The five step cloning process to confine the mutagenesis to the target gene of interest involves: 1) error-prone PCR amplification of the gene of interest; 2) restriction enzyme digestion; 3) gel purification of the desired DNA fragment; 4) ligation into a vector; 5) transformation of the gene variants into a suitable host and screening of the library for improved performance. This method can generate multiple mutations in a single gene simultaneously, which can be useful. A high number of mutants can be generated by EpPCR, so a high-throughput screening assay or a selection method (especially using robotics) is useful to identify those with desirable characteristics.

[00428] Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., Nucleic Acids Res 32:el45 (2004); and Fujii et al, Nat.Protoc. 1 :2493-2497 (2006)) has many of the same elements as 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. Adjusting the Mn ²⁺ concentration can vary the mutation rate somewhat. This technique uses a simple error-prone, single-step method to create a full copy of the plasmid with 3 - 4 mutations/kbp. No restriction enzyme digestion or specific primers are required. Additionally, this method is typically available as a kit.

[00429] DNA or Family Shuffling (Stemmer, W. P. Proc. Natl. Acad. Sci. U.S.A. 91 : 10747- 10751 (1994); and Stemmer, W. P. Nature 370:389-391 (1994)) typically involves digestion of 2 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. Fragments prime each other and recombination occurs when one copy primes another copy (template switch). This method can be used with >lkbp DNA sequences. In addition to mutational recombinants created by fragment reassembly, this method introduces point mutations in the extension steps at a rate similar to error-prone PCR. The method can be used to remove deleterious random neutral mutations that might confer antigenicity.

[00430] Staggered Extension (StEP) (Zhao et al, Nat.Biotechnol 16:258-261 (1998)) 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). Growing fragments anneal to different templates and extend further, which is repeated until full-length sequences are made. Template switching means most resulting fragments have multiple parents. Combinations of low- fidelity polymerases (Taq and Mutazyme) reduce error-prone biases because of opposite mutational spectra.

[00431 ] In Random Priming Recombination (RPR) 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)). Base misincorporation and mispriming via epPCR give point mutations. Short DNA fragments prime one another based on homology and are recombined and reassembled into full-length by repeated thermocycling. Removal of templates prior to this step assures low parental recombinants. This method, like most others, can be performed over multiple iterations to evolve distinct properties. This technology avoids sequence bias, is independent of gene length, and requires very little parent DNA for the application.

[00432] In Heteroduplex Recombination 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)). The mismatch repair step is at least somewhat mutagenic. Heteroduplexes transform more efficiently than linear

homoduplexes. This method is suitable for large genes and whole operons.

[00433] Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al.,

Nat.Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation and size fractionation of ssDNA. Homologous fragments are hybridized in the absence of polymerase to a complementary ssDNA scaffold. Any overlapping unhybridized fragment ends are trimmed down by an exonuclease. Gaps between fragments are filled in, and then ligated to give a pool of full-length diverse strands hybridized to the scaffold (that contains U to preclude amplification). The scaffold then is destroyed and is replaced by a new strand complementary to the diverse strand by PCR amplification. The method involves one strand (scaffold) that is from only one parent while the priming fragments derive from other genes; the parent scaffold is selected against. Thus, no reannealing with parental fragments occurs. Overlapping fragments are trimmed with an exonuclease. Otherwise, this is conceptually similar to DNA shuffling and StEP. Therefore, there should be no siblings, few inactives, and no unshuffled parentals. This technique has advantages in that few or no parental genes are created and many more crossovers can result relative to standard DNA shuffling.

[00434] Recombined Extension on Truncated templates (RETT) 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)). No DNA endonucleases are used. Unidirectional ssDNA is made by DNA polymerase with random primers or serial deletion with exonuclease. Unidirectional ssDNA are only templates and not primers. Random priming and exonucleases don't introduce sequence bias as true of enzymatic cleavage of DNA shuffling/RACHITT. RETT can be easier to optimize than StEP because it uses normal PCR conditions instead of very short extensions. Recombination occurs as a component of the PCR steps—no direct shuffling. This method can also be more random than StEP due to the absence of pauses.

[00435] In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol 352: 191- 204 (2007); Bergquis et al, Biomol.Eng 22:63-72 (2005); Gibbs et al, Gene 271 : 13-20 (2001)) this can be used to control the tendency of other methods such as DNA shuffling to regenerate parental genes. This method can be combined with random mutagenesis (epPCR) of selected gene segments. This can be a good method to block the reformation of parental sequences. No endonucleases are needed. By adjusting input concentrations of segments made, one can bias towards a desired backbone. This method allows DNA shuffling from unrelated parents without restriction enzyme digests and allows a choice of random mutagenesis methods. [00436] Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest.

(Ostermeier et al, Proc Natl Acad Sci U S.A 96:3562-3567 (1999); Ostermeier et al,

Nat.Biotechnol 17: 1205-1209 (1999)). Truncations are introduced in opposite direction on pieces of 2 different genes. These are ligated together and the fusions are cloned. This technique does not require homology between the 2 parental genes. When ITCHY is combined with DNA shuffling, the system is called SCRATCHY (see below). A major advantage of both is no need for homology between parental genes; for example, functional fusions between an E. coli and a human gene were created via ITCHY. When ITCHY libraries are made, all possible crossovers are captured.

[00437] Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) is almost the same as ITCHY except that phosphothioate dNTPs are used to generate truncations. (Lutz, S., M. Ostermeier, and S. J. Benkovic, 2001, Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides. Nucleic Acids Res 29:E16.) Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide more reproducibility, and adjustability.

[00438] SCRATCHY - ITCHY combined with DNA shuffling is a combination of DNA shuffling and ITCHY; therefore, allowing multiple crossovers. (Lutz et al., Proc Natl Acad Sci US.A 98: 1 1248-11253 (2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling. Computational predictions can be used in optimization. SCRATCHY is more effective than DNA shuffling when sequence identity is below 80%.

[00439] In Random Drift Mutagenesis (RNDM) mutations made via epPCR followed by screening/selection for those retaining usable activity. (Bergquist et al., Biomol.Eng 22:63-72 (2005)). Then, these are used in DOGS to generate recombinants with fusions between multiple active mutants or between active mutants and some other desirable parent. Designed to promote isolation of neutral mutations; its purpose is to screen for retained catalytic activity whether or not this activity is higher or lower than in the original gene. RNDM is usable in high throughput assays when screening is capable of detecting activity above background. RNDM has been used as a front end to DOGS in generating diversity. The technique imposes a requirement for activity prior to shuffling or other subsequent steps; neutral drift libraries are indicated to result in higher/quicker improvements in activity from smaller libraries. Though published using epPCR, this could be applied to other large-scale mutagenesis methods.

[00440] Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that: 1) generates pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage; this pool is used as a template to 2) extend in the presence of

"universal" bases such as inosine; 3) replication of a inosine-containing complement gives random base incorporation and, consequently, mutagenesis. (Wong et al., PCR. 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)). Using this technique it can be possible to generate a large library of mutants within 2 -3 days using simple methods. This is very non-directed compared to mutational bias of DNA polymerases. Differences in this approach makes this technique complementary (or alternative) to epPCR.

[00441] In Synthetic Shuffling, overlapping oligonucleotides are designed to encode "all genetic diversity in targets" and allow a very high diversity for the shuffled progeny. (Ness et al., Nat.Biotechnol 20: 1251-1255 (2002)) In this technique, one can design the fragments to be shuffled. This aids in increasing the resulting diversity of the progeny. One can design sequence/codon biases to make more distantly related sequences recombine at rates approaching more closely related sequences and it doesn't require possessing the template genes physically.

[00442] Nucleotide Exchange and Excision Technology NexT 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)). The gene is reassembled using internal PCR primer extension with proofreading polymerase. The sizes for shuffling are directly controllable using varying dUPT::dTTP ratios. This is an end point reaction using simple methods for uracil incorporation and cleavage. One can use other nucleotide analogs such as 8-oxo-guanine with this method. Additionally, the technique works well with very short fragments (86 bp) and has a low error rate. Chemical cleavage of DNA means very few unshuffled clones. [00443] In Sequence Homology-Independent Protein Recombination (SHIPREC) a linker is used to facilitate fusion between 2 distantly/unrelated genes; nuclease treatment is used to generate a range of chimeras between the two. Result is a single crossover library of these fusions. (Sieber et al., Nat.Biotechnol 19:456-460 (2001)). This produces a limited type of shuffling; mutagenesis is a separate process. This technique can create a library of chimeras with varying fractions of each of 2 unrelated parent genes. No homology is needed. SHIPREC was tested with a heme -binding domain of a bacterial CP450 fused to ^-terminal regions of a mammalian CP450; this produced mammalian activity in a more soluble enzyme.

[00444] In Gene Site Saturation Mutagenesis (GSSM) the starting materials are a supercoiled dsDNA plasmid with insert and 2 primers degenerate at the desired site for mutations. (Kretz et al., Methods Enzymol. 388:3-1 1 (2004)). Primers carry the mutation of interest and anneal to the same sequence on opposite strands of DNA; mutation in the middle of the primer and -20 nucleotides of correct sequence flanking on each side. The sequence in the primer is NNN or NNK (coding) and MNN (noncoding) (N = all 4, K = G, T, M = A, C). After extension, Dpnl is used to digest dam-methylated DNA to eliminate the wild-type template. This technique explores all possible amino acid substitutions at a given locus (i.e., one codon). The technique facilitates the generation of all possible replacements at one site with no nonsense codons and equal or near-equal representation of most possible alleles. It does not require prior knowledge of structure, mechanism, or domains of the target enzyme. If followed by shuffling or Gene Reassembly, this technology creates a diverse library of recombinants containing all possible combinations of single-site up-mutations. The utility of this technology combination has been demonstrated for the successful evolution of over 50 different enzymes, and also for more than one property in a given enzyme.

[00445] Combinatorial Cassette Mutagenesis (CCM)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)). Simultaneous substitutions at 2 or 3 sites are possible using this technique. Additionally, the method tests a large multiplicity of possible sequence changes at a limited range of sites. It has been used to explore the information content of lambda repressor DNA-binding domain. [00446] Combinatorial Multiple Cassette Mutagenesis (CMCM)is essentially similar to CCM except it is employed as part of a larger program: 1) Use of epPCR at high mutation rate to 2) ID hot spots and hot regions and then 3) extension by CMCM to cover a defined region of protein sequence space. (Reetz et al, Angew.Chem.Int.Ed Engl. 40:3589-3591 (2001)). As with CCM, this method can test virtually all possible alterations over a target region. If used along with methods to create random mutations and shuffled genes, it provides an excellent means of generating diverse, shuffled proteins. This approach was successful in increasing, by 51 -fold, the enantioselectivity of an enzyme.

[00447] In the Mutator Strains technique conditional is mutator plasmids allow increases of 20- to 4000-X in random and natural mutation frequency during selection and to block accumulation of deleterious mutations when selection is not required. (Selifonova et al., Appl Environ Microbiol 67:3645-3649 (2001)). This technology is based on a plasmid-derived mutD5 gene, which encodes a mutant subunit of DNA polymerase III. This subunit binds to

endogenous DNA polymerase III and compromises the proofreading ability of polymerase III in any of the strain that harbors the plasmid. A broad-spectrum of base substitutions and frameshift mutations occur. In order for effective use, the mutator plasmid should be removed once the desired phenotype is achieved; this is accomplished through a temperature sensitive origin of replication, which allows plasmid curing at 41°C. It should be noted that mutator strains have been explored for quite some time (e.g., see Winter and coworkers, 1996, J. Mol. Biol. 260, 359- 3680. In this technique very high spontaneous mutation rates are observed. The conditional property minimizes non-desired background mutations. This technology could be combined with adaptive evolution to enhance mutagenesis rates and more rapidly achieve desired phenotypes.

[00448] "Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids." (Rajpal et al., Proc Natl Acad Sci U S.A 102:8466-8471 (2005)). Rather than saturating each site with all possible amino acid changes, a set of 9 is chosen to cover the range of amino acid R-group chemistry. Fewer changes per site allows multiple sites to be subjected to this type of mutagenesis. A >800- fold increase in binding affinity for an antibody from low nanomolar to picomolar has been achieved through this method. This is a rational approach to minimize the number of random combinations and should increase the ability to find improved traits by greatly decreasing the numbers of clones to be screened. This has been applied to antibody engineering, specifically to increase the binding affinity and/or reduce dissociation. The technique can be combined with either screens or selections.

[00449] Gene Reassembly is a DNA shuffling method that can be applied to multiple genes at one time or to creating a large library of chimeras (multiple mutations) of a single gene, (on the world-wide web at www.verenium.com/Pages/Technology/EnzymeTech TechEnzyTGR.html) Typically this technology is used in combination with ultra-high-throughput screening to query the represented sequence space for desired improvements. This technique allows multiple gene recombination independent of homology. The exact number and position of cross-over events can be pre-determined using fragments designed via bioinformatic analysis. This technology leads to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Combined with GSSM, a large range of mutations can be tested for improved activity. The method allows "blending" and "fine tuning" of DNA shuffling, e.g. codon usage can be optimized.

[00450] In Silico Protein Design Automation PDA 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. (Hayes et al, Proc Natl Acad Sci U S.A 99: 15926- 15931 (2002)). This technology allows in silico structure-based entropy predictions in order to search for structural tolerance toward protein amino acid variations. Statistical mechanics is applied to calculate coupling interactions at each position - structural tolerance toward amino acid substitution is a measure of coupling. Ultimately, this technology is designed to yield desired modifications of protein properties while maintaining the integrity of structural characteristics. The method computationally assesses and allows filtering of a very large number of possible sequence variants (10 ⁵⁰ ). Choice of sequence variants to test is related to predictions based on most favorable thermodynamics and ostensibly only stability or properties that are linked to stability can be effectively addressed with this technology. The method has been successfully used in some therapeutic proteins, especially in engineering immunoglobulins. In silico predictions avoid testing extraordinarily large numbers of potential variants. Predictions based on existing three-dimensional structures are more likely to succeed than predictions based on hypothetical structures. This technology can readily predict and allow targeted screening of multiple simultaneous mutations, something not possible with purely experimental technologies due to exponential increases in numbers.

[00451] Iterative Saturation Mutagenesis (ISM)involves 1) Use knowledge of

structure/function to choose a likely site for enzyme improvement. 2) Saturation mutagenesis at chosen site using Stratagene QuikChange (or other suitable means). 3) Screen/select for desired properties. 4) With improved clone(s), start over at another site and continue repeating. (Reetz et al, Nat.Protoc. 2:891-903 (2007); and Reetz et al, Angew.Chem.Int.Ed Engl. 45:7745-7751 (2006)). This is a proven methodology assures all possible replacements at a given position are made for screening/selection.

[00452] 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.

[00453] 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 isopropanol, «-butanol, or isobutanol.

[00454] 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 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.

[00455] 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 enable 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. 2002/0168654, WO 2002/055995, and U.S.

2009/0047719.

[00456] 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. 2003/0233218, filed June 14, 2002, and in WO/2003/106998. 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.

[00457] 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.

[00458] 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.

[00459] 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.

[00460] 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.

[00461 ] 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®. [00462] 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.

[00463] 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)).

[00464] 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. [00465] As disclosed herein, a nucleic acid encoding a desired activity of a alternative isopropanol, «-butanol, or isobutanol pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a alternative isopropanol, «-butanol, or isobutanol pathway enzyme or protein to increase production of alternative isopropanol, n- butanol, or isobutanol. 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.

[00466] 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 (¾), 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.

[00467] 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 alternative isopropanol, «-butanol, or isobutanol 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 A cad 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)).

[00468] 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)).

[00469] Further methods include Sequence Homology-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. 388:3-1 1 (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)).

[00470] 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)). [00471 ] 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.

[00472] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

PREPARATION OF AN ISOPROPANOL PRODUCING MICROBIAL ORGANISM HAVING A PATHWAY FOR CONVERTING 4-HYDROXYBUTYRYL-COA TO

ISOPROPANOL

[00473] This example describes the generation of a microbial organism capable of producing isopropanol from 4-hydroxybutyryl-CoA.

[00474] Escherichia coli is used as a target organism to engineer the isopropanol pathway shown in Figure 1 that starts from 4-hydroxybutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing isopropanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

[00475] To generate an E. coli strain engineered to produce 3-hydroxyisobutyric acid, nucleic acids encoding the enzymes utilized in the pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001 ; Ausubel supra, 1999). In particular, the sucD (YP 001396394), 4/zk/ (YP 001396393), bukl (Q45829), and ptb (NP 349676) genes encoding succinic semialdehyde dehydrogenase (CoA-dependent), 4- hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase, and phosphotransbutyrylase activities, respectively, are cloned into an expression vector or integrated into the chromosome as described in Burk et al. (U.S. publication 2009/0075351). In addition, the abfl) (P55792) and crtl (NP 349318.1) genes encoding 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ- isomerase, and enoyl-CoA hydratase activities, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Next, the hbd (NP 349314.1) and atoAD (P76459.1, P76458.1) encoding 3-hydroxybutyryl-CoA dehydrogenase and acetyl- CoA:acetoacetate-CoA transferase activities, respectively, are cloned into the pZS23 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Then, the adc (NP 149328.1) and adh (AAA23199.2) genes encoding acetoacetate decarboxylase and acetone reductase activities are cloned into the pZS13 vector (Expressys, Ruelzheim, Germany) under the

PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well- known molecular biology techniques. The three sets of plasmids are transformed into a 4- hydroxybutyryl-CoA producing strain of E. coli to express the proteins and enzymes required for isopropanol synthesis from 4-hydroxybutyryl-CoA.

[00476] The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). Cobalamin is also supplied to the medium to ensure activity of the mutase enzyme unless the host strain of E. coli is engineered to synthesize cobalamin de novo (see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). The expression of the isopropanol synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce isopropanol is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

[00477] Microbial strains engineered to have a functional isopropanol synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers. [00478] To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also 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 isopropanol. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of isopropanol. Adaptive evolution also can be used to generate better producers of, for example, the 4-hydroxybutyryl-CoA intermediate of the isopropanol product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the isopropanol producer to further increase production.

[00479] For large-scale production of isopropanol, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H ₂ SO ₄ . The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). EXAMPLE II

PREPARATION OF A iV-BUTANOL PRODUCING MICROBIAL ORGANISM HAVING A PATHWAY FOR CONVERTING 4-HYDROXYBUTYRYL-COA TO N-

BUTANOL

[00480] This example describes the generation of a microbial organism capable of producing w-butanol from 4-hydroxybutyryl-CoA.

[00481] Escherichia coli is used as a target organism to engineer the butanol pathway shown in Figure 1 that starts from 4-hydroxybutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing butanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or

microaerobic conditions.

[00482] To generate an E. coli strain engineered to produce 3-hydroxyisobutyric acid, nucleic acids encoding the enzymes utilized in the pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001 ; Ausubel supra, 1999). In particular, the sucD (YP 001396394), 4hbd (YP 001396393), bukl (Q45829), and ptb (NP 349676) genes encoding succinic semialdehyde dehydrogenase (CoA-dependent), 4- hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase, and phosphotransbutyrylase activities, respectively, are cloned into an expression vector or integrated into the chromosome as described in Burk et al. (U.S. publication 2009/0075351). In addition, the abfl) (P55792) gene encoding 4-hydroxybutyryl-CoA dehydratase and vinylacetyl-CoA Δ-isomerase activities is cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Next, the bed NF 349317.1) and etfAB (NP 349315.1, NP 349316.1) genes encoding crotonyl- CoA reductase activity are cloned into the pZS23 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Then, the α/ί (ΑΑΤ66436) and a<a%/ (AAR91477.1) genes encoding butyryl-CoA reductase (aldehyde forming) and butyraldehyde reductase activities are cloned into the pZS13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into a 4-hydroxybutyryl-CoA producing strain of E. coli to express the proteins and enzymes required for butanol synthesis from 4- hydroxybutyryl-CoA.

[00483] The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). Cobalamin is also supplied to the medium to ensure activity of the mutase enzyme unless the host strain of E. coli is engineered to synthesize cobalamin de novo (see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). The expression of the butanol synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce butanol is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

[00484] Microbial strains engineered to have a functional butanol synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

[00485] To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also 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 butanol. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of butanol. Adaptive evolution also can be used to generate better producers of, for example, the 4-hydroxybutyryl-CoA intermediate of the butanol product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al, Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the butanol producer to further increase production.

[00486] For large-scale production of butanol, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE III

PREPARATION OF AN ISOBUTANOL PRODUCING MICROBIAL ORGANISM HAVING A PATHWAY FOR CONVERTING 4-HYDROXYBUTYRYL-COA TO

ISOBUTANOL

[00487] This example describes the generation of a microbial organism capable of producing isobutanol from 4-hydroxybutyryl-CoA.

[00488] Escherichia coli is used as a target organism to engineer the isobutanol pathway shown in Figure 1 that starts from 4-hydroxybutyryl-CoA. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing isobutanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, like ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions. [00489] To generate an E. coli strain engineered to produce 3-hydroxyisobutyric acid, nucleic acids encoding the enzymes utilized in the pathway are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001 ; Ausubel supra, 1999). In particular, the sucD (YP 001396394), 4/zk/ (YP 001396393), bukl (Q45829), and ptb (NP 349676) genes encoding succinic semialdehyde dehydrogenase (CoA-dependent), 4- hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase, and phosphotransbutyrylase activities, respectively, are cloned into an expression vector or integrated into the chromosome as described in Burk et al. (U.S. publication 2009/0075351). In addition, the abfl) (P55792), icm (AAC08713.1), and icmB (CAB59633.1) genes encoding 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ-isomerase, and isobutyryl-CoA mutase activities are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Next, the bed

(NP 349317.1) and etfAB (NP 349315.1, NP 349316.1) genes encoding crotonyl-CoA reductase activity are cloned into the pZS23 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. Then, the adhE (AAV66076.1) gene encoding isobutyryl-CoA reductase (alcohol forming) activity is cloned into the pZS13 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. pZS23 is obtained by replacing the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module by well-known molecular biology techniques. The three sets of plasmids are transformed into a 4- hydroxybutyryl-CoA producing strain of E. coli to express the proteins and enzymes required for isobutanol synthesis from 4-hydroxybutyryl-CoA.

[00490] The resulting genetically engineered organism is cultured in glucose-containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). Cobalamin is also supplied to the medium to ensure activity of the mutase enzyme unless the host strain of E. coli is engineered to synthesize cobalamin de novo (see, for example, Raux et al., J. Bacteriol. 178:753-767 (1996)). The expression of the isobutanol synthesis genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification of mRNA, immunoblotting, and the like. Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individual activities. The ability of the engineered E. coli strain to produce isobutanol is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass spectrometry (LCMS). [00491 ] Microbial strains engineered to have a functional isobutanol synthesis pathway are further augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers.

[00492] To generate better producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also 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 isobutanol. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of isobutanol. Adaptive evolution also can be used to generate better producers of, for example, the 4-hydroxybutyryl-CoA intermediate of the isobutanol product. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the isobutanol producer to further increase production.

[00493] For large-scale production of isobutanol, the above organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing the culture vessel, for example, flasks can be sealed with a septum and crimp-cap. Microaerobic conditions also can be utilized by providing a small hole in the septum for limited aeration. The pH of the medium is maintained at a pH of around 7 by addition of an acid, such as H ₂ SO ₄ . The growth rate is determined by measuring optical density using a spectrophotometer (600 nm) and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu, Columbia MD), for example, using an Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad, Hercules CA), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779 (2005)).

EXAMPLE IV

EXEMPLARY HYDROGENASE AND CO DEHYDROGENASE ENZYMES FOR EXTRACTING REDUCING EQUIVALENTS FROM SYNGAS AND EXEMPLARY

REDUCTIVE TCA CYCLE ENZYMES

[00494] 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.

[00495] 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 Hmicola and Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from Chlorobium Hmicola was cloned and characterized in i. coli (Kanao et al., Eur. J. Biochem. 269:3409-3416 (2002). The C. Hmicola 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 activity 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 Umicola

aclB BAB21375.1 12407235 Chlorobium Umicola

aclA AAM72321.1 21647054 Chlorobium tepidum

aclB AAM72322.1 21647055 Chlorobium tepidum

aclA ABI50076.1 114054981 Balnearium lithotrophicum aclB ΑΒΙ500Ί5Λ 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

SPBC 1703.07 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

[00496] 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. Microbiol. 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 Aq ifex 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).

[00497] 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, MDH1 (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.

[00498] 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. 278:45109-451 16 (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)).

[00499] 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: 11 1-116 (1998)).

[00500] 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:

[00501] 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. Scl. U.S.A. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme from H. thermophilus, encoded by korAB, has been cloned and expressed in i. 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. 2002). The kinetics of C02 fixation of both H. thermophilus OFOR enzymes have been characterized (Yamamoto et al., Extremophiles 14:79-85 (2010)). A C02-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.

[00502] 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. 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 Apel472/Apel473 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. 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 Rhodo spirillum rubrum by sequence homology. A two subunit enzyme has also been identified in Chlorobium tepidum (Eisen et al, PNAS 99(14): 9509-14 (2002)).

[00503] 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, H.G., 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 CC -fixing IDH from Chlorobium limicola and was functionally expressed in i. 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 addition to some other candidates listed below.

[00504] 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-i 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 denitriflcans and Thermocrinis albus.

[00505] Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible isomerization of citrate and iso-citrate via the intermediate cw-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)).

Protein GenBank ID GI Number Organism

Suden 1040 ABB44318.1 78497778 Siilfiirimonas denitrificans

(acnB)

Hydth_0755 AD045152.1 308751669 Hydrogenobacter thermophilus

CT0543 (acn) AAM71785.1 21646475 Chlorobium tepidum

dim 2436 YP 001944436.1 189347907 Chlorobium limicola

dim 0515 ACD89607.1 189340204 Chlorobium limicola

acnA NP 460671.1 16765056 Salmonella typhimurium

acnB NP 459163.1 16763548 Salmonella typhimurium

ACOl AAA34389.1 170982 Saccharomyces cerevisiae

[00506] 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 protects 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, PNAS 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 C02-assimilating directions (Ikeda et al. 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.

15645778

HP 1164 NP 207955.1 Helicobacter pylori

RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

Rnfi: EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

Rnfi EDK33311.1 146346775 Clostridium kluyveri

[00507] 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, H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., 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 £. 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)).

[00508] 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.

[00509] 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.

[00510] 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 et al. 2007). An analogous enzyme is found in Campylobacter jejuni (St et al. 2007). A ferredoxin:NADP ⁺ oxidoreductase enzyme is encoded in the E. coli genome by fpr (Bianchi et al. 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. 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). 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. Protein GenBank ID GI Number Organism

HP 1164 NP 207955.1 15645778 Helicobacter pylori

RPA3954 CAE29395.1 39650872 Rhodopseiidomonas paliistris fpr BAH29712.1 225320633 Hydrogeno bacter

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

RnfC EDK33306.1 146346770 Clostridium kluyveri

RnfD EDK33307.1 146346771 Clostridium kluyveri

RnfG EDK33308.1 146346772 Clostridium kluyveri

Rnfi: EDK33309.1 146346773 Clostridium kluyveri

RnfA EDK33310.1 146346774 Clostridium kluyveri

Rnfi EDK3331 1.1 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

[00511 ] 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. thermophilics 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. 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, 1999). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research

Communications, 192(3): (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7 and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed in the table below.

CcarbDRAFT 5296 ZP 05395295.1 255528511 Clostridium carboxidivorans P7

CcarbDRAFT 1615 ZP 05391615.1 255524662 Clostridium carboxidivorans P7

CcarbDRAFT 1304 ZP 05391304.1 255524347 Clostridium carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermiis

hydrogenoformans

fdxN CAA35699.1 46143 Rhodobacter capsulatiis

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 002801 146.1 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 yf L AP 003148.1 89109368 Escherichia coli K-12

[00512] 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.

[00513] 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 call 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. 2008) and Trypanosoma brucei (Riviere et al. 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. 2008). Similar succinyl-CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al. 2008), Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri (Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoA transferase encoded by peal and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al. 2002). The aforementioned proteins are identified below.

[00514] 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. 1997), Bacillus subtilis, and Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementioned proteins are identified below.

Protein GenBank ID GI Number Organism

OXCT1 NP 000427 4557817 Homo sapiens

OXCT2 NP 071403 1 1545841 Homo sapiens

[00515] 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.

[00516] 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)). Homo logs can be found m Azoarcus sp. T, Aromatoleum aromaticum EbNl, and Geobacter metallireducens GS-15. The aforementioned proteins are identified below.

Protein GenBank ID GI Number Organism

bbsF AAU45406.1 52421825 Azoarcus sp. T

bbsE YP 158075.1 56476486 Aromatoleum aromaticum EbNl bbsF YP 158074.1 56476485 Aromatoleum aromaticum EbNl

Gmet 1521 YP 384480.1 78222733 Geobacter metallirediicens GS-15

Gmet 1522 YP 384481.1 78222734 Geobacter metallirediicens GS-15

[00517] 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.

[00518] 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 i. 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.

citF CAA56217.1 565619 Klebsiella pneumoniae cite CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citC BAH66541.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae

[00519] 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. co /z ^' jt>wr (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)).

[00520] The formation of acetyl-CoA from acetylphosphate is catalyzed by

phosphotransacetylase (EC 2.3.1.8). The pta gene from i. 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

19 A (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 dX., App. Environ. Microbiol. 55:317-322 (1989); Walter et al., Gene 134: 107-11 1 (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).

[00521] 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 AF121 1 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 P. 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.

[00522] The product yields per C-mol of substrate of microbial cells synthesizing reduced fermentation products such as isobutanol, 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 ¾ 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, ¾, 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.

[00523] Here, we show specific examples of how additional redox availability from CO and/or ¾ can improve the yields of reduced products such as isobutanol.

[00524] When both feedstocks of sugar and syngas are available, the syngas components CO and ¾ 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 isobutanol production pathways. Theoretically, all carbons in glucose will be conserved, thus resulting in a maximal theoretical yield to produce isobutanol from glucose.

[00525] As shown in above example, 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 ¾ and CO 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. In case of isobutanol production from glucose or sugar, the theoretical yields improve can be improved under this regime. Such improvements provide environmental and economic benefits and greatly enhance sustainable chemical production.

[00526] Herein below the enzymes and the corresponding genes used for extracting redox from syngas 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 (i.e., CO oxidation).

[00527] In M. 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 metalUreducens GS-15, Chlorobium phaeobacteroides DSM 266, Clostridium cellulolyticum H10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380, and Campylobacter curvus 525.92.

CODH YP 384856.1 78223109 Geobacter metal lireducens GS-15

Cpha266 0148 YP 910642.1 119355998 Chlorobium

(cytochrome c) phaeobacteroides DSM266

Cpha266 0149 YP 910643.1 119355999 Chlorobium

phaeobacteroides DSM266 (CODH)

Ccel 0438 YP 002504800.1 220927891 Clostridium cellulolyticum H10

Ddes 0382 YP 002478973.1 220903661 Desulfovibrio desulfuricans subsp. (CODH) desulfuricans sir. ATCC 27774

Ddes 0381 YP 002478972.1 220903660 Desulfovibrio desulfuricans subsp. (CooC) desulfuricans sir. ATCC 27774

Pear 0057 YP 355490.1 7791767 Pelobacter carbinolicus DSM

2380

(CODH)

Pear 0058 YP 355491.1 7791766 Pelobacter carbinolicus DSM

2380

(CooC)

Pear 0058 YP 355492.1 7791765 Pelobacter carbinolicus DSM

2380

(HypA)

CooS (CODH) YP 001407343.1 154175407 Campylobacter curvus 525.92

[00528] 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. I) hydrogenoformans

CooF YP 360645 78044791 Carboxydothermiis

hydrogenoformans

HypA YP 360646 78044340 Carboxydothermiis

hydrogenoformans

CooH YP 360647 78043871 Carboxydothermiis

hydrogenoformans

CooU YP 360648 78044023 Carboxydothermiis

hydrogenoformans

CooX YP 360649 78043124 Carboxydothermiis

hydrogenoformans

CooL YP 360650 78043938 Carboxydothermiis

hydrogenoformans

CooK YP 360651 78044700 Carboxydothermiis

hydrogenoformans

CooM YP 360652 78043942 Carboxydothermiis

hydrogenoformans

CooC YP 360654.1 78043296 Carboxydothermiis

hydrogenoformans

CooA-1 YP 360655.1 78044021 Carboxydothermiis

hydrogenoformans

CooL AAC451 18 1515468 Rhodospirilliim rubriim

CooX AAC45119 1515469 Rhodospirilliim rubriim

CooU AAC45120 1515470 Rhodospirilliim rubriim

CooH AAC45121 1498746 Rhodospirilliim rubriim

CooF AAC45122 1498747 Rhodospirilliim rubriim

CODH (CooS) AAC45123 1498748 Rhodospirilliim rubriim

CooC AAC45124 1498749 Rhodospirilliim rubriim

CooT AAC45125 1498750 Rhodospirilliim rubriim

CooJ AAC45126 1498751 Rhodospirilliim rubriim

[00529] 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.J Biochem. 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., How E. coli is equipped to oxidize hydrogen under different redox conditions, J Biol Chem published online Nov 16, 2009). 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-H4folate reductase.

[00530] 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

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

[00531] 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 2A). 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.

[00532] Proteins in M. thermoacetica whose genes are homologous to the E. coli hyp genes are shown below.

[00533] Proteins in M. thermoacetica that are homologous to the E. coli Hydrogenase 3 and/or 4 proteins are listed in the following table.

[00534] In addition, several gene clusters encoding hydrogenase functionality are present in M. thermoacetica and their corresponding protein sequences are provided below.

Moth 1 194 YP 430051 83590042 Moorella thermoacetica

Moth 1 195 YP 430052 83590043 Moorella thermoacetica

Moth 1 196 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

[00535] Ralstonia eutropha H16 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 02-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 of the Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)). 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

HoxlE AAP50519.1 37787351 Thiocapsa roseopersicina

HoxlF AAP50520.1 37787352 Thiocapsa roseopersicina

HoxlU AAP50521.1 37787353 Thiocapsa roseopersicina

HoxlY AAP50522.1 37787354 Thiocapsa roseopersicina

HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina

[00536] 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.

[00537] 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).

[00538] 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, PCKl, 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 C02-fixing enzyme following adaptive evolution (Zhang et al. 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)), Anaerobiospirillum succiniciproducens

(Laivenieks et ah, 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.

[00539] 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)). Protein GenBank ID GI Number Organism

PYC1 NP 011453 6321376 Saccharomyces cerevisiae

PYC2 NP 009777 6319695 Saccharomyces cerevisiae

Pyc YP 890857.1 1 18470447 Mycobacterium smegmatis

[00540] 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 fro Ascaris suum 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)).

[00541] 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.

[00542] 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 ¾, as disclosed herein, improve the yields of isobutanol when utilizing carbohydrate-based feedstock. For example, isobutanol can be produced from succinyl-CoA via pathways in Figures 2 and 3. Exemplary enzymes for the conversion succinyl-CoA to isobutanol include all those shown in Figures 2 and 3.

[00543] 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. For example, isobutanol can be produced from the glycolysis intermediate, pyruvate. Exemplary enzymes for the conversion of pyruvate to isobutanol include the enzymes recited in Figure 2B.

EXAMPLE V

METHODS FOR HANDLING CO AND ANAEROBIC CULTURES

[00544] This example describes methods used in handling CO and anaerobic cultures.

[00545] 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. Therefore, 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.

[00546] 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.

[00547] B. Handling of CO in larger quantities fed to large-scale cultures. Fermentation cultures are fed either CO or a mixture of CO and ¾ 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.

[00548] 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.

[00549] 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.

[00550] 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.

[00551] 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 ( iCl 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- 1 OOOx 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- 1 -thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs.

[00552] 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

[00553] This example describes assay methods for measuring CO oxidation (CO

dehydrogenase; CODH).

[00554] 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 ~l/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 M. 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 rubriim, Moorella thermoacetica and Desulfitobacterium hafniense.

[00555] 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.

[00556] 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. [00557] 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 of M. 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.

[00558] 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 mL 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.

[00559] Mta98/Mta99 are E. coli MG1655 strains that express methanol methyltransferase genes from M. thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M. thermoacetica CODH operons.

[00560] 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 M. 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.

[00561] 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 4. 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.

[00562] 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 5. 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.

[00563] 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)

[00564] This example describes the tolerance of E. coli for high concentrations of CO.

[00565] 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 [00566] 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.

[00567] 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

[00568] 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.

[00569] 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

[00570] This example describes the use of carboxylic acid reductases (CAR) to carry out the conversion of a carboxylic acid to an aldehyde.

[00571] Any intermediate carboxylic acid in a isobutanol pathway (or accessible carboxylic acid via its CoA derivative) can be converted to an aldehyde, if so desired. 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 i. 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 Industries, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, FL. (2006)).

[00572] Additional car and npt genes can be identified based on sequence homology. Gene Accession No. GI No. Organism

fadD9 YP 978699.1 121638475 Mycobacterium bovis BCG

BCG 2812c YP 978898.1 121638674 Mycobacterium bovis BCG nfa20150 YP 1 18225.1 54023983 Nocardia farcinica IFM 10152 nfa40540 YP 120266.1 54026024 Nocardia farcinica IFM 10152

Streptomyces griseus subsp.

SGR 6790 YP 001828302.1 182440583 griseus NBRC 13350

Streptomyces griseus subsp.

SGR 665 YP 001822177.1 182434458 griseus NBRC 13350

[00573] 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.

Tsukamiirella

TpauDRAFT 33060 ZP 04027864.1 227980601 paurometabola DSM

20162

Tsukamiirella

TpauDRAFT 20920 ZP 04026660.1 227979396 paurometabola DSM

20162

CPCC7001 1320 ZP 05045132.1 254431429 Cyanobium PCC7001

Dictyostelium discoideum

DDBDRAFT 0187729 XP 636931.1 66806417

AX4

[00574] 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 i. 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.

[00575]

[00576] Cloning and Expression of Carboxylic Acid Reductase. Escherichia coli is used as a target organism to engineer the pathway for isobutanol. E. coli provides a good host for generating a non-naturally occurring microorganism capable of producing isobutanol. E. coli is amenable to genetic manipulation and is known to be capable of producing various intermediates and products effectively under various oxygenation conditions.

[00577] To generate a microbial organism strain such as an E. coli strain engineered to produce isobutanol, 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.

[00578] The car gene (GNM 720) was cloned by PCR from Nocardia genomic DNA. Its nucleic acid and protein sequences are shown in Figures 12A and 12B, 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 13 A and 13B, 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 14, 15, and 16, respectively. The plasmids are transformed into a host cell to express the proteins and enzymes required for isobutanol production.

[00579] Additional CAR variants were generated. A codon optimized version of CAR 891 was generated and designated 891 GA. The nucleic acid and amino acid sequences of CAR 891GA are shown in Figures 17A and 17B, 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; T941 S; 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.

[00580] The CAR variants were screened for activity, and numerous CAR variants were found to exhibit CAR activity. This example describes the use of CAR for converting carboxylic acids to aldehydes.

SEQUENCE LISTING

[00581] The present specification is being filed with a computer readable form (CRF) copy of the Sequence Listing. The CRF entitled 12956-142 SEQLIST.txt, which was created on June 17, 2012 and is 77,801 bytes in size, is identical to the paper copy of the Sequence Listing and is incorporated herein by reference in its entirety. [00582] 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 and embodiments provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.