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Schizochytrium Vs Shewanella

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Schizochytrium Vs Shewanella US 2008.0026440A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0026440 A1 Metz et al. (43) Pub. Date: Jan. 31, 2008 (54) PUFA POLYKETIDE SYNTHASE SYSTEMS (60) Provisional application No. 60/284.066, filed on Apr. AND USES THEREOF 16, 2001. Provisional application No. 60/298,796, filed on Jun. 15, 2001. Provisional application No. (75) Inventors: James G. Metz, Longmont, CO (US); 60/323.269, filed on Sep. 18, 2001. James H. Flatt, Colorado Springs, CO (US); Jerry M. Kuner, Longmont, CO Publication Classification (US); William R. Barclay, Boulder, (51) Int. Cl. CO (US) CI2P 7/64 (2006.01) C7H 2L/00 (2006.01) Correspondence Address: CI2N I/00 (2006.01) SHERIDAN ROSS PC CI2N I/2 (2006.01) 1560 BROADWAY CI2N 5/04 (2006.01) SUTE 12OO (52) U.S. Cl. ...................... 435/134: 435/243; 435/252.3: DENVER, CO 80202 435/419; 536/23.2 (73) Assignee: MARTEK BIOSCIENCES CORPO (57) ABSTRACT RATION, Columbia, MD (US) The invention generally relates to polyunsaturated fatty acid (21) Appl. No.: 11/689,608 (PUFA) polyketide synthase (PKS) systems isolated from or derived from non-bacterial organisms, to homologues (22) Filed: Mar. 22, 2007 thereof, to isolated nucleic acid molecules and recombinant nucleic acid molecules encoding biologically active Related U.S. Application Data domains of such a PUFA PKS system, to genetically modi fied organisms comprising PUFA PKS systems, to methods (63) Continuation of application No. 10/124,800, filed on of making and using Such systems for the production of Apr. 16, 2002, now Pat. No. 7,247.461, and which is bioactive molecules of interest, and to novel methods for a continuation-in-part of application No. 09/231,899, identifying new bacterial and non-bacterial microorganisms filed on Jan. 14, 1999, now Pat. No. 6,566,583. having such a PUFA PKS system. Comparison of PKS Orfs/domains: Schizochytrium vs Shewanella Orf6 Orf 7 Of 8 AI-S-S- DH OH ER - 2 2 V Of B DH DH ER Of C Amino acid sequence homology = 20-35% = 35-50% > 50% Patent Application Publication Jan. 31, 2008 Sheet 1 of 3 US 2008/0026440 A1 É N O er S g 2 CN (N f \d \d want g 2 Patent Application Publication Jan. 31, 2008 Sheet 2 of 3 US 2008/0026440 A1 Z'OIH Patent Application Publication Jan. 31, 2008 Sheet 3 of 3 US 2008/0026440 A1 unuifi?oo2|qøSSA(OZIL)oorsow SHRIOSXAVIQ?umpul??oo2??oç (SJ)IO9,8.Iedas) JOVJIO8!4 ~~~~.No. US 2008/0026440 A1 Jan. 31, 2008 PUFA POLYKETDE SYNTHASE SYSTEMIS AND 0005. In contrast to the Type I and II systems, in modular USES THEREOF PKS systems, each enzyme domain is used only once in the production of the end product. The domains are found in CROSS-REFERENCE TO RELATED very large proteins and the product of each reaction is passed APPLICATIONS on to another domain in the PKS protein. Additionally, in all 0001. This application is a continuation of U.S. applica of the PKS systems described above, if a carbon-carbon tion Ser. No. 10/124,800, filed Apr. 16, 2002, entitled “PUFA double bond is incorporated into the end product, it is always Polyketide Synthase Systems and Uses Thereof.” which in the trans configuration. claims the benefit of priority under 35 U.S.C. S 119(e) to: 0006. In the Type I and Type II PKS systems described U.S. Provisional Application Ser. No. 60/284,066, filed Apr. above, the same set of reactions is carried out in each cycle 16, 2001, entitled “A Polyketide Synthase System and Uses until the end product is obtained. There is no allowance for Thereof: U.S. Provisional Application Ser. No. 60/298.796, the introduction of unique reactions during the biosynthetic filed Jun. 15, 2001, entitled “A Polyketide Synthase System procedure. The modular PKS systems require huge proteins and Uses Thereof; and U.S. Provisional Application Ser. that do not utilize the economy of iterative reactions (i.e., a No. 60/323,269, filed Sep. 18, 2001, entitled “Thraus distinct domain is required for each reaction). Additionally, tochytrium PUFA PKS System and Uses Thereof. This as stated above, carbon-carbon double bonds are introduced application is also a continuation-in-part of copending U.S. in the trans configuration in all of the previously described application Ser. No. 09/231,899, filed Jan. 14, 1999, entitled PKS systems. “Schizochytrium PKS Genes”. Each of the above-identified patent applications is incorporated herein by reference in its 0007 Polyunsaturated fatty acids (PUFAs) are critical entirety. components of membrane lipids in most eukaryotes (Lau ritzen et al., Prog. Lipid Res. 401 (2001); McConn et al., 0002 This application does not claim the benefit of Plant J. 15, 521 (1998)) and are precursors of certain priority from U.S. application Ser. No. 09/090,793, filed Jun. hormones and signaling molecules (Heller et al., Drugs 55. 4, 1998, now U.S. Pat. No. 6,140,486, although U.S. appli 487 (1998); Creelman et al., Annu. Rev. Plant Physiol. Plant cation Ser. No. 09/090,793 is incorporated herein by refer Mol. Biol. 48, 355 (1997)). Known pathways of PUFA ence in its entirety. synthesis involve the processing of saturated 16:0 or 18:0 FIELD OF THE INVENTION fatty acids (the abbreviation X;Y indicates an acyl group containing X carbon atoms and Y cis double bonds; double 0003. This invention relates to polyunsaturated fatty acid bond positions of PUFAs are indicated relative to the methyl (PUFA) polyketide synthase (PKS) systems from microor carbon of the fatty acid chain (c)3 or co6) with systematic ganisms, including eukaryotic organisms, such as Thraus methylene interruption of the double bonds) derived from tochytrid microorganisms. More particularly, this invention fatty acid synthase (FAS) by elongation and aerobic desatu relates to nucleic acids encoding non-bacterial PUFA PKS ration reactions (Sprecher, Curr. Opin. Clin. Nutr. Metab. systems, to non-bacterial PUFA PKS systems, to genetically Care 2, 135 (1999); Parker-Barnes et al., Proc. Natl. Acad. modified organisms comprising non-bacterial PUFA PKS Sci. USA 97,8284 (2000); Shanklin et al., Annu. Rev. Plant systems, and to methods of making and using the non Physiol. Plant Nol. Biol. 49, 611 (1998)). Starting from bacterial PUFA PKS systems disclosed herein. This inven acetyl-CoA, the synthesis of DHA requires approximately tion also relates to a method to identify bacterial and 30 distinct enzyme activities and nearly 70 reactions includ non-bacterial microorganisms comprising PUFA PKS sys ing the four repetitive steps of the fatty acid synthesis cycle. temS. Polyketide synthases (PKSs) carry out some of the same BACKGROUND OF THE INVENTION reactions as FAS (Hopwood et al., Annu. Rev. Genet. 24, 37 (1990); Bentley et al., Annu. Rev. Microbiol. 53,411 (1999)) 0004 Polyketide synthase (PKS) systems are generally and use the same Small protein (or domain), acyl carrier known in the art as enzyme complexes derived from fatty protein (ACP), as a covalent attachment site for the growing acid synthase (FAS) systems, but which are often highly carbon chain. However, in these enzyme systems, the com modified to produce specialized products that typically show plete cycle of reduction, dehydration and reduction seen in little resemblance to fatty acids. Researchers have attempted FAS is often abbreviated so that a highly derivatized carbon to exploit polyketide synthase (PKS) systems that have been chain is produced, typically containing many keto- and described in the literature as falling into one of three basic types, typically referred to as: Type II, Type I and modular. hydroxy-groups as well as carbon-carbon double bonds in The Type II system is characterized by separable proteins, the trans configuration. The linear products of PKSs are each of which carries out a distinct enzymatic reaction. The often cyclized to form complex biochemicals that include enzymes work in concert to produce the end product and antibiotics and many other secondary products (Hopwood et each individual enzyme of the system typically participates al., (1990) supra; Bentley et al., (1999), supra; Keating et al., several times in the production of the end product. This type Curr. Opin. Chem. Biol. 3, 598 (1999)). of system operates in a manner analogous to the fatty acid 0008 Very long chain PUFAs such as docosahexaenoic synthase (FAS) systems found in plants and bacteria. Type acid (DHA; 22:6c)3) and eicosapentaenoic acid (EPA: I PKS systems are similar to the Type II system in that the 20:5c)3) have been reported from several species of marine enzymes are used in an iterative fashion to produce the end bacteria, including Shewanella sp (Nichols et al., Curr. Op. product. The Type I differs from Type II in that enzymatic Biotechnol. 10, 240 (1999); Yazawa, Lipids 31, S (1996): activities, instead of being associated with separable pro DeLong et al., Appl. Environ. Microbiol. 51, 730 (1986)). teins, occur as domains of larger proteins. This system is Analysis of a genomic fragment (cloned as plasmid pEPA) analogous to the Type I FAS systems found in animals and from Shewanella sp. strain SCRC2738 led to the identifi fungi. cation of five open reading frames (Orfs), totaling 20 Kb. US 2008/0026440 A1 Jan. 31, 2008 that are necessary and sufficient for EPA production in E. Synthesis of EPA was found only in the high-speed pellet coli (Yazawa, (1996), supra). Several of the predicted pro fraction, indicating that EPA synthesis can occur without tein domains were homologues of FAS enzymes, while other reliance on enzymes of the E. coli FAS or on soluble regions showed no homology to proteins of known function.
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