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2012/058686 A2 © (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 v - n 3 May 2012 (03.05.2012) 2012/058686 A2 (51) International Patent Classification: Way, Berkeley, California 94703 (US). ZHANG, Jing- C12N 9/10 (2006.01) wei [CN/US]; 734 Bush Street, Apartment 22, San Fran cisco, California 94108 (US). ZOTCHEV, Sergey [RU/ (21) International Application Number: NO]; Klostergata 40A, N-7030 Trondheim (NO). PCT/US201 1/058660 (74) Agents: LOCKYER, Jean M. et al; Kilpatrick (22) International Filing Date: Townsend & Stockton LLP, Two Embarcadero Center, 31 October 201 1 (3 1.10.201 1) 8th Floor, San Francisco, California 941 11 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Langi English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 61/408,4 11 29 October 2010 (29.10.2010) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (71) Applicant (for all designated States except US): THE HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, REGENTS OF THE UNIVERSITY OF CALIFOR¬ KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, NIA [US/US]; 1111 Franklin Street, 12th Floor, Oakland, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, California 94607 (US). NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, (72) Inventors; and TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (75) Inventors/ Applicants (for US only): FORTMAN, Jef¬ ZM, ZW. frey L. [US/US]; 1534 Innes Avenue, San Francisco, Cal ifornia 94124 (US). HAGEN, Andrew [US/US]; 2417 (84) Designated States (unless otherwise indicated, for every 8th Street, Berkeley, California 94710 (US). KATZ, kind of regional protection available): ARIPO (BW, GH, Leonard [US/US]; 6389 Longcroft Drive, Oakland, Cali GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, fornia 9461 1 (US). KEASLING, Jay [US/US]; 1630 UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, Sterling Avenue, Berkeley, California 94708 (US). RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, IS, POUST, Sean [US/US]; 1907 1/2 Martin Luther King DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IT, [Continued on next page] (54) Title: HYBRID POLYKETIDE SYNTHASES < 00 00 2-Mcthylhcxancdioic acid Adipic acid Figure 7 © (57) Abstract: The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd- o chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured pro duces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin. w o 2012/058686 A2 1I 11 II I 1 III I lll ll M III III II I II LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, Published: SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, ·,; · · ; ; without international search report and to b e republishedv GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). t t t nR e ' upon recei p t oj t h a t report ( u l 48.2(g)) Hybrid Polyketide Synthases CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims benefit of priority to U.S. provisional application no. 61/408,41 , filed October 29, 2010, which application is herein incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT |0002j This invention was made with government support under Contract No. DE-AC02- 05CH1 1231 awarded by the U.S. Department of Energy and Award No. 0540879 awarded by the National Science Foundation. The government has certain rights in the invention FIELD OF THE INVENTION [0003] This invention relates to the production of useful compounds with polyketide synthases. BACKGROUND OF THE INVENTION [0004] Dicarboxylic acids (diacids) are important compounds that are used in the manufacture of commercial polymers (e.g. polyesters, polyurethanes). The use of hybrid polyketide synthases to produce diacids having a carbon backbone with an odd number of carbon atoms is disclosed in International Patent Application Publication No. WO 2009/121066. However, commercial polymers are typically produced from reactants derived from petroleum and petrochemicals. [0005] For example, as illustrated in Figure , the dicarboxylic acid adipic acid [1] is used mainly as a monomer in the production of nylon 6,6 [2], a polyamide generated through the reaction of [1] with hexane-l,6-diamine. Polyesters (for use in fabrics and plastics of many compositions) are formed through the polymerization of terephthalic acid [3] and a dialcohol (diol) such as ethylene glycol (to make polyethylene terephthalate [4]), propane diol (poly(l,3-propanediol terephthalate) [5]) or butanediol (poly(l,4-butanediolphthalate) [6]. Adipic acid is also used in the synthesis of various polyesters. Currently adipic acid is synthesized via oxidation of cyclohexane and similar petrochemicals using traditional chemical synthesis. [0006| Lactams are important compounds useful in the manufacture of a variety of compounds, including commercial polymers, particularly polyamides such as Nylon 6 and Nylon 12. Caprolactam is the sole source of Nylon 6, which is used in the production of durable fibers for carpets and in other products. Larger chain lactams, such as Nylon 12, are used in engineering plastics where their physical properties make them more desirable. The open chain form of these molecules are also accessible using the technology described herein. These cognate acids are also used in many of the same applications. For example, 6- aminohexanoic acid is the open chain form of caprolactam and can also be polymerized to produce Nylon 6. [0007] Diamines are used extensively in the production of polymers, predominantly Nylons, as described above. 10008] The large scale worldwide use of nylons and polyesters requires the production of millions of metric tons of adipic acid, caprolactam and 1,6-hexanediamine annually. The diacids, lactams, and diamines are themselves synthesized from starting materials extracted from petroleum. There is a need for new methods to synthesize such compounds in a manner that reduces dependence on oil. [0009] Complex polyketides comprise a large class of natural products that are synthesized in bacteria (mainly members actinomycete family; e.g. Streptomyces), fungi and plants. Polyketides form the aglycone component of a large number of clinically important drugs, such as antibiotics (e.g. erythromycin, tylosin), antifungal agents (e.g. nystatin), anticancer agents (e.g. epothilone), immunosuppressives (e.g. rapamycin), etc. Though these compounds do not resemble each other either in their structure or their mode of action, they share a common basis for their biosynthesis, which is carried out by a group of enzymes designated polyketide synthases. Polyketide synthases (PKS) employ short chain fatty acyl CoAs in Claisen condensation reactions to produce polyketides. Unlike fatty acid synthases, which utilize acetyl CoA as the starter and malonyl CoA as the extender units and which use a single module iteratively to produce the nascent acyl chains, PKSs are composed of multiple, discrete modules, each catalyzing the chain growth of a single step. The present invention provides methods and compositions for employing polyketide synthases to produce diacids, lactams, diamines, e.g., for the production of commercial polymers. BRIEF SUMMARY OF THE INVENTION [0010] The present invention provides for a polyketide synthase (PKS), and/or optionally non-ribosomal peptide synthase and/or CoA ligase, capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The PKS is not a naturally occurring PKS. In some embodiments of the invention, the even-chain or odd-chain diacid or lactam or diamine is not a compound synthesized by a naturally occurring PKS. In some embodiments of the invention, the PKS is a hybrid PKS comprising modules, domains, and/or portions thereof from two or more naturally occurring PKSs. [001 ] The present invention also provides for a recombinant nucleic acid that encodes a polyketide synthase (PKS) of the present invention. The recombinant nucleic acid can be replicon capable of stable maintenance in a host cell. In some embodiments, the replicon is stably integrated into a chromosome of the host cell. In some embodiments, the replicon is a plasmid. The present invention also provides for a vector or expression vector comprising a recombinant nucleic acid of the present invention. The present invention additionally provides for a host cell comprising any of the recombinant nucleic acid and/or PKS of the present invention. In some embodiments, the host cell, when cultured under a suitable condition, is capable of producing an even- or odd-chain diacid, lactam, or diamine. [0012] The present invention provides a method of producing an even-chain or odd-chain diacid or lactam or diamine, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the even-chain or odd-chain diacid or lactam or diamine is produced. [0013] The present invention provides for a composition comprising an even-chain or odd- chain diacid or lactam or diamine isolated from a host cell from which the even-chain or odd- chain diacid or lactam or diamine was produced, and trace residues and/or contaminants of the host cell.
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