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(12) United States Patent (10) Patent No.: US 9,334,514 B2 Fortman Et Al

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(12) United States Patent (10) Patent No.: US 9,334,514 B2 Fortman Et Al USOO9334514B2 (12) United States Patent (10) Patent No.: US 9,334,514 B2 Fortman et al. (45) Date of Patent: May 10, 2016 (54) HYBRID POLYKETIDE SYNTHASES (52) U.S. Cl. CPC ............... CI2P 17/186 (2013.01); CI2N 15/52 (75) Inventors: Jeffrey L. Fortman, San Francisco, CA (2013.01); CI2P 7/44 (2013.01); C12P 7/46 (US); Andrew Hagen, Berkeley, CA (2013.01); C12P 13/001 (2013.01): CI2P (US); Leonard Katz, Oakland, CA 13/005 (2013.01): CI2P 13/04 (2013.01): CI2P (US); Jay D. Keasling, Berkeley, CA 17/10 (2013.01) (US): Sean Poust, Berkeley, CA (US); (58) Field of Classification Search Jingwei Zhang, San Francisco, CA None (US); Sergey Zotchev, Trondheim (NO) See application file for complete search history. (73) Assignee: The Regents of the University of (56) References Cited California, Oakland, CA (US) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 8,569,023 B2 * 10/2013 Katz et al. ..................... 435,142 U.S.C. 154(b) by 0 days 2013/0267012 A1* 10/2013 Steen et al. .............. 435/254.21 (21) Appl. No.: 13A882,099 FOREIGN PATENT DOCUMENTS y x- - - 9 WO WO 2009.113853 A2 9, 2009 (22) PCT Fled: Oct. 31, 2011 WO WO 2009/121066 * 10/2009 WO WO 2009/121066 A1 10, 2009 (86). PCT No.: PCT/US2O11?058660 OTHER PUBLICATIONS S371 (c)(1), (2), (4) Date: Jul. 10, 2013 Holtzel et al., Spirofungin, a New Antifungal Antibiotic from Streptomyces violaceusniger Ti 4113. The Journal of Antibiotics (87) PCT Pub. No.: WO2012/058686 (1998), vol. 51, No. 8, pp. 699-707.* PCT Pub. Date: May 3, 2012 (Continued) (65) Prior Publication Data Primary Examiner — Alexander Kim US 2013/0280766A1 Oct. 24, 2013 (74) Attorney, Agent, or Firm — Kilpatrick Townsend & s Stockton LLP Related U.S. Application Data (57) ABSTRACT (60) gypal application No. 61/408,411, filed on Oct. The present invention provides for a polyketide synthase s (PKS) capable of synthesizing an even-chain or odd-chain (51) Int. Cl. diacid or lactam or diamine. The present invention also pro CI2P 17/18 (2006.01) vides for a host cell comprising the PKS and when cultured CI2P 7/44 (2006.015 produces the even-chain diacid, odd-chain diacid, or KAPA. CI2P 7/46 (2006.01) The present invention also provides for a host cell comprising CI2PI3/00 (2006.015 the PKS capable of synthesizing a pimelic acid or KAPA, and CI2PI3/04 (2006.01) when cultured produces biotin. (Continued) 17 Claims, 20 Drawing Sheets () KS (5) O O -O adipic acid US 9,334,514 B2 Page 2 (51) Int. Cl. Jez et al.: “Expanding the biosynthetic repertoire of plant type III CI2N 15/52 polyketide synthases by altering starter molecule specificity; Proc. (2006.01) Natl. Acad. Sci. USA;99(8):5319-5324 (2002). CI2P I 7/10 (2006.01) Khosla et al.; "Tolerance and specificity of polyketide synthases”; Ann. Rev. Biochem.: 68:219-253 (1999). (56) References Cited Li et al., “Automated genome mining for natural products’; BMC Bioinformatics; 10(185): 1-10 (2009). Marsden et al., “Engineering broader specificity into an antibiotic OTHER PUBLICATIONS producing polyketide synthase'; Science; 279(5348): 199-202 (1998). International Search Report from PCT/US2011/058660, dated May May et al.; “Crystal structure of DhbE, an archetype for aryl acid 25, 2012. activating domains of modular nonribosomal peptide synthases”; Proc. Natl. Acad. Sci. USA; 99(19): 12120-12125 (2002). Ansarietal.; “NRPS-PKS: a knowledge-based resource for analysis Menche et al.; "Stereochemical determination and complex of NRPS/PKS megasynthases”: Nucl. Acids Res. 32(Web Server biosynthetic assembly of etnangien, a highly potent RNA polymerase issue):W405-413 (Jul. 1, 2004). inhibitor from the myxobacterium Sorangium cellulosum'. J. Am. Brautaset et al.; “Biosynthesis of the polyene antifungal antibiotic Chem. Soc.: 130(43): 14234-14243 (2008). nystatin in Streptomyces noursei ATCC 11455: analysis of the gene Oliynyk et al., “Complete genome sequence of the erythromycin cluster and deduction of the biosynthetic pathway': Chem. Biol. producing bacterium Saccharopolyspora erythraeaNRRL233338'; 7(6):395-403 (2000). Nat. Biotechnol. 25(4):447-453 (2007). Challis et al., “Mining microbial genomes for new natural products Rachidet al.: “Unusual chemistry in the biosynthesis of the antibiotic and biosynthetic pathways'; Microbiology; 154(6): 1555-1569 chondrochlorens”; Chem. Biol.; 16(1):70-81 (2009). Reeves et al.; "Alteration of the Substrate specificity of modular (2008). polyketide synthase acyltransferase domain through site-specific Challis et al., “Predictive, structure-based model fo amino acid rec mutations”; Biochemistry, 40(51): 15464-15470 (2001). ognition by nonribosomal peptide synthetase adenylation domains'; Staunton et al.; “Polyketide biosynthesis: a millenium review; Nat. Chem. Biol.; 7(3):2111-224 (2000). Prod. Rep.; 18(4):380-416 (2001) ePub Jun. 4, 2001. Dutton et al.: “Novel avermectins produced by mutational Wiesmann et al.; “Polyketide synthesis in vitro on a modular biosynthesis”; J. Antibiot. (Tokyo); 44(3):357-365 (1991). polyketide synthase': Chem. Biol. 2(9):583-589 (1995). Finking et al.; "Biosynthesis of nonribosomal peptides'; Annu. Rev. Microbiol.; 58:453-488 (2004). * cited by examiner U.S. Patent May 10, 2016 Sheet 1 of 20 US 9,334,514 B2 Figure 1 U.S. Patent May 10, 2016 Sheet 2 of 20 US 9,334,514 B2 Loading Modules Extender Modules G l X 1. RN-S R > R ^ R n O O M N O p U.S. Patent May 10, 2016 Sheet 3 of 20 US 9,334,514 B2 N 1N1-1- Ni O O O O P- - HO Novel polyamide O > HO (Cir -OH O Schlin(Cir ga Novel polyester. --- O HO O Novel polyamide O > O O. Hos Chin -OH --- O (CH)n e Novel polyester Figure 3 U.S. Patent May 10, 2016 Sheet 4 of 20 US 9,334,514 B2 2-methylhexanoic acid Figure 5 U.S. Patent May 10, 2016 Sheet 5 of 20 US 9,334,514 B2 Domain Function 1 CoA Binding 2 CoA ligase 3 ATP grasp 4 ATP grasp 5 CoA ligase Figure 6 A. B 2-Methylhexanedioic acid Adipic acid Figure 7 U.S. Patent May 10, 2016 Sheet 6 of 20 US 9,334,514 B2 E. L. Succinate loading Trans-AT from Ent E. M. Nys 15 DEBSTE S S O O T O H B d O H Figure 8 Figure 9 U.S. Patent May 10, 2016 Sheet 7 of 20 US 9,334,514 B2 O -N Pinelic acid HO Y (CH2)5cooH ATP, CoA Pimelyl-CoA synthetase - ON Pimeloyl-CoA CoAm S (CH2)5COOH 'N 8-amino-7-oxononanoate synthase H2N Ol pelargonic7-keto 8-amino acid (KAPA) H3C (CH2)5COOH SAM, re- DAPA synthase H2N1 NNH2 acid7,8-diamino-pelargonic (DAPA) H3C (CH2)5COOH ATP, CO2 Dethiobiotin synthase O H N NH Dethiobiotin H3C (CH2)5COOH Biotin synthase -R XX Biotin S (CH2)4-COOH Figure 10 U.S. Patent May 10, 2016 Sheet 8 of 20 US 9,334,514 B2 adipic acid Figure 11 U.S. Patent May 10, 2016 Sheet 9 of 20 US 9,334,514 B2 HO. O. O O 2-2 2 OH O a'ala 2- -OH e-CCC's HO 1 2 Figure 12 Voce die 12 {) / O TE (PKS) ALDH (SpiF) ADH (SpE W FMospih) --EST (Spin) es -- Propionic acid C) O W A. O "Prespirofungin" Figure 13 U.S. Patent May 10, 2016 Sheet 10 of 20 US 9,334,514 B2 - MOdule 1 - M - MOdule 3-TE OH O OH Release and Modification - s O s& ADPCACD ProPIONCAClo Figure 14 U.S. Patent May 10, 2016 Sheet 11 of 20 US 9,334,514 B2 O O ----' sco Malonyl-CoA O S-COA Succinyl-CoA Figure 15 U.S. Patent May 10, 2016 Sheet 12 of 20 US 9,334,514 B2 OH O O O He O O N-W O H Adipic Acid Figure 16 Module 1-TE O C O O -e- O O N -1 O Adipic Acid Figure 17 U.S. Patent May 10, 2016 Sheet 13 of 20 US 9,334,514 B2 GuCOSe Y SCCA S O Ya O O O -e Acetyl-CoA -> O O O H Malonyl-CoA Maoric Acid Figure 18 II.1.ii.b. Design 2. GUCOSe S-COA (O. O O Acetyl-CoA -> O O O H H Malonyl-CoA MaoriCACid Figure 19 U.S. Patent May 10, 2016 Sheet 14 of 20 US 9,334,514 B2 28 : S O O HO to-cO O Figure 20 Load Mode 1 2 roA 4-guanidinobutanamide 4-aminobutyryl-CoA : NH HN 4-aloitatarie lsNH 2 N arginine H Figure 21 U.S. Patent May 10, 2016 Sheet 15 of 20 US 9,334,514 B2 s s us-- O K s'-- Ivriz. t - U () N Gu o i. th O NH ----N-1N- Or O * N O : r" - " - E.so r Er HN O. N O O--A- bio- * fo-A axisn HN O HO-A-r-SN HN O Ho oh Butrosin B Figure 22 U.S. Patent May 10, 2016 Sheet 16 of 20 US 9,334,514 B2 Load Module 1 Caprolactam H CO2 b -H Glutamate NH Figure 23 U.S. Patent May 10, 2016 Sheet 17 of 20 US 9,334,514 B2 Load Module 1 Module 2 a N-1 Caprolactam H2 b N - H c Figure 24 U.S. Patent May 10, 2016 Sheet 18 of 20 US 9,334,514 B2 A load Module 1 O N-1 H Aminocaproic Acid B load Woe O t -- N-1 H H Aminocaproic Acid C load Module Mode 2 H h Aminocaproic Acid Figure 25 U.S.
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