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WO 2010/127303 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 4 November 2010 (04.11.2010) WO 2010/127303 Al (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12P 7/16 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/US2010/033271 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 30 April 2010 (30.04.2010) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (25) Filing Language: English TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 61/174,482 30 April 2009 (30.04.2009) US GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): GENO- TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, MATICA, INC. [US/US]; 10520 Wateridge Circle, San ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Diego, CA 92121 (US). MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventor; and ML, MR, NE, SN, TD, TG). (75) Inventor/Applicant (for US only): BURGARD, Antho¬ ny, P. [US/US]; 10520 Wateridge Circle, San Diego, CA Declarations under Rule 4.17: 9212 1 (US). — as to applicant's entitlement to apply for and be granted (74) Agents: BEHAR, Victor et al; Mcdermott, Will & a patent (Rule 4.1 7(U)) Emery LLP, 11682 El Camino Real, Suite 400, San Published: Diego, CA 92130-2047 (US). — with international search report (Art. 21(3)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: ORGANISMS FOR THE PRODUCTION OF ISOPROP ANOL, N-BUTANOL, AND ISOBUTANOL (57) Abstract: A non-naturally occurring microbial organism having an isopropanol pathway includes at least one exogenous nu cleic acid encoding an isopropanol pathway enzyme. The pathway includes an enzyme selected from a 4-hydroxybutyryl-CoA de hydratase, a crotonase, a 3 -hydro xybutyryl- CoA dehydrogenase, an acetoacetyl-CoA synthetase, an acetyl-CoA:acetoacetate- CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase, and an acetone reductase. A non-naturally occur ring microbial organism having an w-butanol pathway includes at least one exogenous nucleic acid encoding an w-butanol path way enzyme. Other non- naturally occurring microbial organism have w-butanol or isobutanol pathways. The organisms are cul tured to produce isopropanol, w-butanol, or isobutanol. ORGANISMS FOR THE PRODUCTION OF ISOPROPANOL. /i-BUTANOL. AND ISOBUTANOL STATEMENT OF RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Application No. 61/174,482, filed April 30, 2009, the entire contents of which are incorporated herein by reference. BACKGROUND OF THE INVENTION 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. 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 b y product 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. 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 butylacrylate 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. 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 w-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)). 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 OF THE INVENTION 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. 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. In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a w-butanol pathway having at least one exogenous nucleic acid encoding a w-butanol pathway enzyme expressed in a sufficient amount to produce w-butanol. The w-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). In some embodiments, the present invention provides a method for producing w-butanol comprising culturing a non-naturally occurring microbial organism having an w-butanol pathway, under conditions and for a sufficient period of time to produce w-butanol. 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). In some embodiments, the present invention
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