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Ep 1623008 B1 (19) TZZ_ ¥ZZ_T (11) EP 1 623 008 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A01H 9/00 (2006.01) 30.07.2014 Bulletin 2014/31 (86) International application number: (21) Application number: 04758405.7 PCT/US2004/009323 (22) Date of filing: 26.03.2004 (87) International publication number: WO 2004/087879 (14.10.2004 Gazette 2004/42) (54) PUFA POLYKETIDE SYNTHASE SYSTEMS AND USES THEREOF PUFA-POLYKETIDSYNTHASE-SYSTEME UND VERWENDUNGEN DAVON SYSTEMES DE POLYCETIDES SYNTHASE D’ACIDE GRAS POLYINSATURE ET LEURS UTILISATIONS (84) Designated Contracting States: • FAN K W ET AL: "Eicosapentaenoic and AT BE BG CH CY CZ DE DK EE ES FI FR GB GR docosahexaenoic acids production by and okara- HU IE IT LI LU MC NL PL PT RO SE SI SK TR utilizing potential of thraustochytrids" JOURNAL OF INDUSTRIAL MICROBIOLOGY AND (30) Priority: 26.03.2003 US 457979 P BIOTECHNOLOGY, BASINGSTOKE, GB, vol. 27, no.4, 1 October 2001 (2001-10-01), pages 199-202, (43) Date of publication of application: XP002393382 ISSN: 1367-5435 08.02.2006 Bulletin 2006/06 • METZ J G ET AL: "Production of polyunsaturated fatty acids by polyketide synthases in both (73) Proprietor: DSM IP Assets B.V. prokaryotes and eukaryotes" SCIENCE, 6411 TE Heerlen (NL) AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, (72) Inventors: WASHINGTON, DC, vol. 293, 13 July 2001 • METZ, James G. (2001-07-13), pages 290-293, XP002956819 ISSN: Longmont, CO 80501 (US) 0036-8075 • WEAVER, Craig A. • NAPIER J A: "Plumbing the depths of PUFA Boulder, CO 80301 (US) biosynthesis: a novel polyketide synthase-like • BARCLAY, William R. pathway from marine organisms" TRENDS IN Boulder, CO 80303 (US) PLANTSCIENCE, ELSEVIER SCIENCE, OXFORD, • FLATT, James H. GB, vol. 7, no. 2, 1 February 2002 (2002-02-01), Longmont, CO 80503 (US) pages 51-54, XP002956820 ISSN: 1360-1385 • QIU X ET AL: "Identification of a Delta 4 fatty acid (74) Representative: Mallalieu, Catherine Louise et al desaturase from Thraustochytrium sp. involved D Young & Co LLP in the biosynthesis of docosahexanoic acid by 120 Holborn heterologous expression in Saccharomyces London EC1N 2DY (GB) cerevisiae and Brassica juncea." THE JOURNAL OF BIOLOGICAL CHEMISTRY 24 AUG 2001, vol. (56) References cited: 276, no. 34, 24 August 2001 (2001-08-24), pages WO-A-00/42195 WO-A-02/083870 31561-31566, XP002522023 ISSN: 0021-9258 US-A- 5 908 622 US-A- 6 140 486 • METZ ET AL.: ’Production of polyunsaturated US-A1- 2002 138 874 US-A1- 2002 156 254 fatty acids by polyketide synthase in both US-A1- 2002 194 641 prokaryotes and eukaryotes’ SCIENCE vol. 293, 13 July 2001, pages 290 - 293, XP002956819 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 623 008 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 623 008 B1 • QUI ET AL.: ’Identification of a delta4 fatty acid • WEETE ET AL.: ’Lipids and ultrastructure of desaturase from Thraustochytrium sp. involved Thrauchytrium sp. ATCC26185’ AM. OIL CHEM. in the biosynthesis’ J. BIOL. CHEM. vol. 276, no. SOC. vol. 32, no. 8, 1997, pages 839 - 845, 34, 24 August 2001, pages 31561 - 31566, XP009077836 XP002522023 • SINGH ET AL.: ’Microbial production of • YOKOCHI ET AL.: ’Optimization of docosahexaenoic acid (DHA, C22: 6)’ ADV. APPL. docosahexaenoic acid production’ APPL. MICROBIOL. vol. 45, 1997, pages 271 - 312, MICROBIOL. BIOTECHNOL. vol. 49, 1998, pages XP008046097 72 - 76, XP002928890 • WATANABE ET AL.: ’Fatty acid synthesis of an eicosapentaenoic acid-producing bacterium: de novo synthesis, chain elongation, and desaturation system’ J. BIOCHEM. vol. 122, 1997, pages 467 - 473, XP008107154 • NAKAHARA ET AL.: ’Production of docosahexaenoic and docosapentaenoic acids by Schizochytrium sp. isolated from Yap islands’ J.AM. OIL CHEM. SOC. vol. 73, no. 11, 1996, pages 1421 - 1426, XP000891671 2 EP 1 623 008 B1 Description Field of the Invention 5 [0001] This invention relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from microor- ganisms, including eukaryotic organisms, such as Thraustochytrid microorganisms. More particularly, this invention relates to nucleic acids encoding non-bacterial PUFA PKS systems, to non-bacterial PUFA PKS systems, to genetically modified organisms comprising non-bacterial PUFA PKS systems, and to methods of making and using the non-bacterial PUFA PKS systems disclosed herein. This invention also relates to genetically modified microorganisms and methods 10 to efficiently produce lipids (triacylglyerols (TAG), as well as membrane-associated phospholipids (PL)) enriched in various polyunsaturated fatty acids (PUFAs) and particularly, eicosapentaenoic acid (C20:5, ω-3; EPA) by manipulation of a PUFA polyketide synthase (PKS) system. Background of the Invention 15 [0002] Polyketide synthase (PKS) systems are generally known in the art as enzyme complexes derived from fatty acid synthase (FAS) systems, but which are often highly modified to produce specialized products that typically show little resemblance to fatty acids. It has now been shown, however, that polyketide synthase systems exist in marine bacteria and certain microalgae that are capable of synthesizing PUFAs from malonyl-CoA. The PKS pathways for PUFA 20 synthesis in Shewanella and another marine bacteria, Vibrio marinus, are described in detail in U.S. Patent No. 6,140,486. The PKS pathways for PUFA synthesis in the eukaryotic Thraustochytrid, Schizochytrium is described in detail in U.S. Patent 6,566,583. Finally, the PKS pathways for PUFA synthesis in eukaryotes such as members of Thraustochytriales, including the complete structural description of the PUFA PKS pathway in Schizochytrium and the identification of the PUFA PKS pathway in Thraustochytrium, including details regarding uses of these pathways, are described in detail in 25 U.S. Patent Application Publication No. 20020194641, published December 19, 2002 (corresponding to U.S. Patent Application Serial No. 10/124,800, filed April 16, 2002). [0003] Researchers have attempted to exploit polyketide synthase (PKS) systems that have been described in the literature as falling into one of three basic types, typically referred to as: Type II, Type I and modular. The Type II system is characterized by separable proteins, each of which carries out a distinct enzymatic reaction. The enzymes work in 30 concert to produce the end product and each individual enzyme of the system typically participates several times in the production of the end product. This type of system operates in a manner analogous to the fatty acid synthase (FAS) systems found in plants and bacteria. Type I PKS systems are similar to the Type II system in that the enzymes are used in an iterative fashion to produce the end product. The Type I differs from Type II in that enzymatic activities, instead ofbeing associated with separable proteins, occur as domains of larger proteins. This system is analogous to the Type 35 I FAS systems found in animals and fungi. [0004] In contrast to the Type I and II systems, in modular PKS systems, each enzyme domain is used only once in the production of the end product. The domains are found in very large proteins and the product of each reaction is passed on to another domain in the PKS protein. Additionally, in all of the PKS systems described above, if a carbon- carbon double bond is incorporated into the end product, it is always in the trans configuration. 40 [0005] In the Type I and Type II PKS systems described above, the same set of reactions is carried out in each cycle until the end product is obtained. There is no allowance for the introduction of unique reactions during the biosynthetic procedure. The modular PKS systems require huge proteins that do not utilize the economy of iterative reactions (i.e., a distinct domain is required for each reaction). Additionally, as stated above, carbon-carbon double bonds are introduced in the trans configuration in all of the previously described PKS systems. 45 [0006] Polyunsaturated fatty acids (PUFAs) are critical components of membrane lipids in most eukaryotes (Lauritzen et al., Prog. Lipid Res. 40 1 (2001); McConn et al., Plant J. 15, 521 (1998)) and are precursors of certain hormones and signaling molecules (Heller et al., Drugs 55, 487 (1998); Creelman et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355 (1997)). Known pathways of PUFA synthesis involve the processing of saturated 16:0 or 18:0 fatty acids (the abbreviation X:Y indicates an acyl group containing X carbon atoms and Y double bonds (usually cis in PUFAs); double- 50 bond positions of PUFAs are indicated relative to the methyl carbon of the fatty acid chain ω(3 or ω6) with systematic methylene interruption of the double bonds) derived from fatty acid synthase (FAS) by elongation and aerobic desaturation reactions (Sprecher, Curr. Opin. Clin. Nutr. Metab. Care 2, 135 (1999); Parker-Barnes et al., Proc. Natl. Acad. Sci. USA 97, 8284 (2000); Shanklin et al., Annu. Rev. Plant Physiol. Plant Nol. Biol. 49, 611 (1998)). Starting from acetyl-CoA, the synthesis of docosahexaenoic acid (DHA) requires approximately 30 distinct enzyme activities and nearly 70 reactions 55 including the four repetitive steps of the fatty acid synthesis cycle. Polyketide synthases (PKSs) carry out some of the same reactions as FAS (Hopwood et al., Annu. Rev. Genet. 24, 37 (1990); Bentley et al., Annu. Rev. Microbiol. 53, 411 (1999)) and use the same small protein (or domain), acyl carrier protein (ACP), as a covalent attachment site for the growing carbon chain.
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