USOO7645597B2 (12) United States Patent (10) Patent No.: US 7,645,597 B2 Metz et al. (45) Date of Patent: Jan. 12, 2010 (54) PUFA POLYKETIDE SYNTHASE SYSTEMS CI2N 5/82 (2006.01) AND USES THEREOF CI2P 7/64 (2006.01) (52) U.S. Cl. ....................... 435/134:435/419,435/189: (75) Inventors: James G. Metz, Longmont, CO (US); 435/252.3; 435/320.1; 435/410,536/23.2 Craig A. Weaver, Boulder, CO (US); (58) Field of Classification Search ....................... None William R. Barclay, Boulder, CO (US); See application file for complete search history. James H. Flatt, Colorado Springs, CO (US) (56) References Cited (73) Assignee: Martek Biosciences Corporation, U.S. PATENT DOCUMENTS Columbia, MD (US) 5,130,242 A 7/1992 Barclay et al. (*) Notice: Subject to any disclaimer, the term of this inued patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 11/778,608 CA 252O795 10, 2004 (22) Filed: Jul. 16, 2007 (Continued) (65) Prior Publication Data OTHER PUBLICATIONS US 2008/OO44871 A1 Feb. 21, 2008 Abbadi et al., Eur, J. Lipid Sci. Technol. 103: 106-113 (2001). Related U.S. Application Data (Continued) (60) Division of application No. 1 1/676.971, filed on Feb. Primary Examiner Anand U Desai 20, 2007, which is a division of application No. Assistant Examiner William W. Moore 10/810,352, filed on Mar. 26, 2004, now Pat. No. (74) Attorney, Agent, or Firm—Sheridan Ross P.C. 7.211,418, and a continuation-in-part of application No. 10/124,800, filed on Apr. 16, 2002, now Pat. No. (57) ABSTRACT 7.247.461, and a continuation-in-part of application No. 09/231,899, filed on Jan. 14, 1999, now Pat. No. The invention generally relates to polyunsaturated fatty acid 6,566,583. (PUFA) polyketide synthase (PKS) systems, to homologues (60) Provisional application No. 60/457,979, filed on Mar thereof, to isolated nucleic acid molecules and recombinant 26, 2003, provisional appl ication No. 60,284.066. nucleic acid molecules encoding biologically active domains filed O Apr. 16, 2001, provisional application No. of Such a PUFA PKS system, to genetically modified organ 60/298.796, filed on Jun. 15, 2001, provisional appli- isms comprising PUFA PKS systems, to methods of making cation No. 60,323,269, filedons ep 18, 2001 and using Such systems for the production of bioactive mol s1- Y - s • Y-s ecules of interest, and to novel methods for identifying new (51) Int. Cl. bacterial and non-bacterial microorganisms having Such a CI2N 15/53 (2006.01) PUFA PKS system. CI2N 15/74 (2006.01) CI2N 15/79 (2006.01) 21 Claims, 3 Drawing Sheets Orf A Orf B KS CLF AT ER Orf C 166 kDa US 7,645,597 B2 Page 2 U.S. PATENT DOCUMENTS WO WO 2004/087879 10, 2004 WO WO 2006/008099 1, 2006 5,246,841 A 9, 1993 Yazawa et al. WO WO 2006/034228 3, 2006 5,310,242 A 5, 1994 Golder 5,639,790 A 6, 1997 Voelker et al. OTHER PUBLICATIONS 5,672.491 A 9, 1997 Khosia et al. 5,683,898 A 11/1997 Yazawa et al. Allen et al., Appl. Envir. Microbiol. 65(4): 1710-1720 (1999). 5,798,259 A 8, 1998 Yazawa et al. Bateman et al., Nucl. Acids Res., 30(1):276-280 (2002). 5,908,622 A 6/1999 Barclay Bentley et al., Annu. Rev. Microbiol., 53:411-46 (1999). 6,033,883. A 3, 2000 Barr et al. Bisang et al., Nature, 401:502-505 (1999). 6,140,486 A 10, 2000 Facciotti et al. Bork, TIG, 12(10):425-427 (1996). 6,503,706 B1 1/2003. Abken et al. Brenner, TIG, 15(4): 132-133 (1999). 6,566,583 B1 5, 2003 Facciotti et al. Broun et al., Science, 282:1315-1317 (1998). 7,001,772 B2 2/2006 Roessler et al. Creelman et al., Annu. Rev. Plan Physiol. Plant Mol. Biol. 48:355-81 7,125,672 B2 10/2006 Picataggio et al. (1997). 7,211,418 B2 5, 2007 Metz et al. DeLong & Yayanos, Appl. Environ. Microbiol. 51(4):730-737 7,214,853 B2 5, 2007 Facciotti et al. (1986). 7,217,856 B2 5, 2007 Weaver et al. Doerks, TIG, 14(6):248-250 (1998). 7,256,022 B2 8, 2007 Metz et al. Facciotti et al., “Cloning and Characterization of Polyunsaturated 7,256,023 B2 8, 2007 Metz et al. Fatty Acids (PUFA) Genes from Marine Bacteria” in Proceedings of 7,259,295 B2 8, 2007 Metz et al. the international Symposium on progress and prospect of marine 7,271,315 B2 9, 2007 Metz et al. biotechnology (China Ocean Pres 1999), pp. 404-405 Abstract. 2002/0138874 A1 9/2002 Mukerji et al. Heath et al., J. Biol. Chem., 271 (44):27795-27801 (1996). 2002/0156254 A1 10/2002 Qiu et al. Hopwood & Sherman, Annu. Rev. Genet. 24:37-66 (1990). 2002/0194641 A1 12/2002 Metz et al. Hutchinson, Annu. Rev. Microbiol. 49:201-238 (1995). 2004/OOO5672 A1 1/2004 Santi et al. Jostensen & Landfald, FEMS Microbiology Letters, 151:95-101 2004/0010817 A1 1/2004 Shockey et al. (1997). 2004/O139498 A1 7/2004 Jaworski et al. Katz & Donadio, Annu. Rev. Microbiol., 47:875-912 (1993). 2004/0172682 A1 9/2004 Kinney et al. Kealing et al., Curr. Opin. Chem. Biol. 3:598-606 (1999). 2005, OO14231 A1 1/2005 Mukerji et al. Kyle et al., HortScience, 25:1523-26 (1990). 2005.0089865 A1 4/2005 Napier et al. Magnuson, Microbil. Rev. 57(3):522-542 (1993) Abstract. 2005, 0164.192 A1 7/2005 Graham et al. Metz et al., Science, 293:290-293 (2001). 2007/0244192 A1 10, 2007 Metz Nakahara, Yukagaku, 44(10):821-7 (1995). 2007/0245431 A1 10, 2007 Metz et al. Nasu et al., J. Ferment. Bioeng, 122:467-473 (1997). 2007,0256146 A1 11, 2007 Metz et al. Nichols et al., Curr. Opin Biotechnol., 10:240-246 (1999). 2007/0270494 A1 11, 2007 Metz et al. Nogi et al., Extremophiles, 2:1-7 (1998). 2008/0022422 A1 1/2008 Weaver et al. Parker-Barnes et al., PNAS,97(15):8284-8289 (2000). 2008/0026434 A1 1/2008 Weaver et al. Sánchez et al., Chemistry & Biolosy, 8:725-738 (2001). 2008/0026435 A1 1/2008 Weaver et al. Shanklin et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-41 2008, 0026438 A1 1/2008 Metz et al. (1998). 2008, 0026439 A1 1/2008 Metz et al. Smith et al., Nature Biotechnol. 15:1222-1223 (1997). 2008, OO323510026440 A1,A1 2,1/2008 2008 Metz et al. Somervilleille Am. J. Clin. Nutt,Nutr., 58(2 supp):270S-275Ssupp) (1993) 2008, OO38378 A1 2/2008 Metz et al. Van de Loo, Proc. Natl. Acad. Sci. USA, 92:6743-6747 (1995). 2008.OO38379 A1 2/2008 Metz et al. Watanabe et al., J. Biochem., 122:467-473 (1997). 2008.OO38790 A1 2/2008 Metz et al. Yalpaniet al., The Plant Cell, 13:1401-1409 (2001). 2008.OO38792 A1 2/2008 Metz et al. Metz et al. Production of Polyunsaturated Fatty Acids by Polyketide 2008.OO38793 A1 2/2008 Metz et al. Synthase in Both Prokaryotes and Eukaryotes. Science Jul. 12, 2001, 2008, OO38794 A1 2/2008 Metz et al. vol. 293, pp. 290-293. 2008.OO38795 A1 2/2008 Metz et al. Qiu et al. Identification of a delta4 fatty acid desaturase from 2008, OO387.96 A1 2/2008 Metz et al. Thraustochytrium sp. involved in the biosynthesis. J. Biol. Chem. 2008, OO387.97 A1 2/2008 Metz et al. Aug. 24, 2001, vol. 276, No. 34, pp. 31561-31566. 2008/0040822 A1 2/2008 Metz et al. Yokochi et al. Optimization of docosahexaenoic acid production. 2008/0044867 A1 2/2008 Metz et al. App. Microbiol. Biotechnol. 1998, vol. 49, pp. 72-76. 2008/0044868 A1 2/2008 Metz et al. Nakahara et al. Production of docosahexaenoic and 2008/0044869 A1 2/2008 Metz et al. docosapentaenoic acids by Schizochytrium sp. isolated from Yap 2008/0044870 A1 2/2008 Metz et al. Islands. 1996 J. Am. Oil Chem. Soc. 1996, vol. 73, No. 11, pp. 2008/0044872 A1 2/2008 Metz et al. 1421-1426. 2008/0044873 A1 2/2008 Metz et al. Weete et al. Lipids and Ultrasctructure of Thrauchytrium sp. 2008, OOSO790 A1 2/2008 Metz et al. ATCC26.185. 1997, Am Oil Chem. Soc. vol. 32, No. 8, pp. 839-845. 2008. O148433 A1 6, 2008 Metz et al. Singh et al. Microbial Production of Docosahexaenoic Acid (DHA. C22:6). Adv. Appl. Microbial, 1997. vol. 45, pp. 271-312. FOREIGN PATENT DOCUMENTS Napier, Trends Plant Sci. Feb. 2002; 7(2): 51-4. International Search Report for International (PCT) Patent Applica EP O594.868 5, 1994 tion No. PCT/US04/09323, mailed Apr. 4, 2007. EP O823475 2, 1998 Written Opinion for International (PCT) Patent Application No. WO 9.323545 11, 1993 PCT/US04/09323, mailed Apr. 4, 2007. WO 962.1735 T 1996 International Preliminary Report on Patentability for International WO 98.46764 10, 1998 (PCT) Patent Application No. PCT/US04/09323, mailed May 9, WO 98.55625 12/1998 2007. WO OO42.195 T 2000 Examiner's First Report for Australian Patent Application No. WO WO O2/O83870 10, 2002 2004225485, mailed Nov. 17, 2006. US 7,645,597 B2 Page 3 First Examination Report for Indian Patent Application No. 4359. Wallis et al., “Polyunsaturated fatty acid synthesis: what will they DELNP/2005, dated Mar.