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WO 2012/129450 Al 27 September 2012 (27.09.2012) P O P C T

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WO 2012/129450 Al 27 September 2012 (27.09.2012) P O P C T (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 WO 2012/129450 Al 27 September 2012 (27.09.2012) P O P C T (51) International Patent Classification: (74) Agents: WILLIS, Michael, A. et al; Wilson Sonsini CUP 1/00 (2006.01) Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA 94304-1050 (US). (21) International Application Number: PCT/US20 12/030209 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) Date: International Filing AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 22 March 2012 (22.03.2012) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (25) Filing Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, (26) Publication Language: English KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/466,363 22 March 201 1 (22.03.201 1) US OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, 61/466,433 22 March 201 1 (22.03.201 1) US SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/539,378 26 September 201 1 (26.09.201 1) us TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 61/539,162 26 September 201 1 (26.09.201 1) us (84) Designated States (unless otherwise indicated, for every (71) Applicant (for all designated States except US): OPX BI¬ kind of regional protection available): ARIPO (BW, GH, OTECHNOLOGIES, INC. [US/US]; 2425 55th Street, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Suite 100, Boulder, CO 80301 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (72) Inventors; and DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (75) Inventors/Applicants (for US only): LYNCH, Michael, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, D. [US/US]; 2425 55th Street, Suite 100, Boulder, CO SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, 80301 (US). LIPSCOMB, Tanya, E.w. [US/US]; 2425 GW, ML, MR, NE, SN, TD, TG). 55th Street, Suite 100, Boulder, CO 80301 (US). TRA- HAN, Ashley, D. [US/US]; 2425 55th Street, Suite 100, Published: Boulder, CO 80301 (US). SINGH, Amar [IN/US]; 2425 — with international search report (Art. 21(3)) 55th Street, Suite 100, Boulder, CO 80301 (US). — before the expiration of the time limit for amending the WOLTER, Travis [US/US]; 2425 55th Street, Suite 100, claims and to be republished in the event of receipt of Boulder, CO 80301 (US). amendments (Rule 48.2(h)) — with sequence listing part of description (Rule 5.2(a)) © (54) Title: MICROBIAL PRODUCTION OF CHEMICAL PRODUCTS AND RELATED COMPOSITIONS, METHODS AND SYSTEMS (57) Abstract: Metabolically engineered microorganism strains are disclosed, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a chemical product. Such chemical products include polyketides, 3-hydroxypropionic o acid, and various other chemical products described herein. Methods of production also may be applied to further downstream products, such as consumer products. In various embodiments, modifications to a microorganism and/or culture system divert, at least transiently, usage of malonyl-coA from the fatty acid biosynthesis pathway and thereby provides for usage of the malonyl-coA o for a chemical product other than a fatty acid. In various embodiments, the fatty acid biosynthesis pathway is modulated to produce specific fatty acids or combinations of fatty acids. MICROBIAL PRODUCTION OF CHEMICAL PRODUCTS AND RELATED COMPOSITIONS, METHODS AND SYSTEMS RELATED APPLICATIONS [0001] This application claims priority to U.S. provisional patent applications US 61/466,363, filed on March 22, 201 1; US 61/466,433, filed on March 22, 201 1; US 61/539,162, filed September 26, 201 1; and US 61/539,378, filed September 26, 201 1; each of which are hereby incorporated by reference in their entirety. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] This invention was made with Government support under DE-AR0000088 awarded by the United States Department of Energy. The Government has certain rights in this invention. SEQUENCE LISTINGS [0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 22, 2012, is named 34246760.txt and is 848 kbytes in size. INCORPORATION BY REFERENCE [0004] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BACKGROUND OF THE INVENTION [0005] There is increased emphasis on renewable production of fuels and industrial chemicals as spikes in petroleum costs occur and as petroleum finite resources are consumed. Notwithstanding advances in the general field, there remains a need to find metabolic solutions to renewable production so as to increase rate, productivity, yield, and overall cost-effectiveness for biosynthesis of various chemical products. SUMMARY OF THE INVENTION [0006] Disclosed herein is a method for producing a chemical, the method comprising combining a carbon source, a microorganism, and a cell culture to produce the chemical, wherein a) said cell culture comprises an inhibitor of fatty acid synthase and/or the microorganism is genetically modified for reduced enzymatic activity in at least two of the microorganism's fatty acid synthase pathway enzymes, providing for reduced conversion of malonyl-CoA to fatty acids; and b) the microorganism additionally has one or more genetic modifications relating to a metabolic production pathway from malonyl-CoA to the chemical. In various embodiments, the at least two fatty acid synthase pathway enzymes with reduced enzymatic activity are an enoyl-coA reductase and a beta-ketoacyl-ACP synthase. In one example, the enoyl-coA reductase is fabl or a peptide of 80% or more homology to SEQ ID NO: 14. In addition, the beta-ketoacyl-ACP synthase may be selected from the group consisting of fabB, fabF, a peptide of 80% or more homology to SEQ ID NO: 9, and a peptide of 80%> or more homology to SEQ ID NO: 8. In various emdodiments, a third fatty acid synthase pathway enzyme is modified for reduced enzymatic activity. For example, the enoyl-coA reductase is fabl or a peptide of 80% or more homology to SEQ ID NO: 14, the beta-ketoacyl-ACP synthase is fabB or a peptide of 80% homology or more to SEQ ID NO: 9, and the third fatty acid synthase pathway enzyme is fabF or a peptide of 80% homology or more to SEQ ID NO: 8. [0007] In various embodiments, the third fatty acid synthase pathway enzyme is a malonyl-coA- ACP transacylase. For example, the malonyl-coA-ACP transacylase is modified fabD or a peptide of 80% or more homology to SEQ ID NO: 7. In various embodiments, the enoyl-coA reductase is fabl or a peptide of 80% or more homology to SEQ ID NO: 14, the beta-ketoacyl- ACP synthase is selected from the group consisting of fabB or a peptide of 80%> homology or more to SEQ ID NO: 9, and fabF or a peptide of 80% homology or more to SEQ ID NO: 8, and the malonyl-coA-ACP transacylase is fabD or a peptide of 80% or more homology to SEQ ID NO: 7. [0008] In various emdodiments, the at least two fatty acid synthase pathway enzymes with reduced enzymatic activity are an enoyl-coA reductase and a malonyl-coA-ACP transacylase. For example, the enoyl-coA reductase is fabl or a peptide of 80% or more homology to SEQ ID NO: 14. As an additional example, the malonyl-coA-ACP transacylase is fabD or a peptide of 80% or more homology to SEQ ID NO: 7. [0009] Also disclosed are methods as described above, where the microorganism is E. coli. In any of the embodiments described herein, the method may be performed at above room temperature. Preferably, the method is performed at a temperature between 25°C and 50°C. [0010] Also disclosed are methods as described herein, where the microorganism has one or more genetic modifications to increase levels of pantothenate. As an example, the one or more genetic modifications to increase levels of pantothenate is a modification of panE or a peptide of 80% or more homology to panE. [0011] In various embodiments, the microorganism has one or more genetic modifications to increase pyruvate dehydrogenase activity. For example, the one or more genetic modifications to increase pyruvate dehydrogenase activity is a modification of aceE or a peptide of 80% or more homology to SEQ ID NO: 172. [0012] In various embodiments, the increased pyruvate dehydrogenase activity is resistant to inhibition by elevated NADH levels. In addition, the microorganism may be further genetically modified to have reduced alcohol dehydrogenase activity. As an example, the microorganism is genetically modified to have a modification of adhE or a peptide of 80% or more homology to adhE. [0013] Also disclosed are methods as described herein, wherein the microorganism is further genetically modified to have one or more genetic modifications relating to a metabolic production pathway from malonyl-CoA to the chemical, wherein the pathway uses NADH as a reducing agent and wherein there is a mutation or deletion of an alcohol dehydrogenase gene.
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